Crystalline d-isoglutamyl-d-tryptophan and monoammonium salt of d-isogutamyl-d-tryptophan

FIELD: chemistry.

SUBSTANCE: disclosed is a method of producing pure crystalline D-isoglutamyl-D-trytophan which involves a step of removing protection from essentially pure N-tert-butoxycarbonyl-D-isoglutamyl-D-tryptophan or diester thereof to yield essentially pure D-isoglutamyl-D- tryptophan. An amorphous ammonium alt of D-isoglutamyl-D- tryptophan (1:1) is also disclosed. Also disclosed is a method of producing a pure monoammonium salt of D-isoglutamyl-D-tryptophan from essentially pure N-tert-butoxycarbonyl-D- isoglutamyl-D-tryptophan. Disclosed is a compound H-D-Glu-(γ-D-Trp-OR2)-α-OR1 and pharmaceutically acceptable acid addition salts thereof. Disclosed is a solid pharmaceutical composition and use thereof as an immunodepressant or anti-psoriasis agent.

EFFECT: improved method.

51 cl, 14 ex, 8 dwg, 1 tbl

 

The technical FIELD

This invention relates to new stable crystalline form D-isoglutamine-D-tryptophan and the way it captures in its pure form, free from inorganic salts. This invention also relates to new stable ammonium salt of D-isoglutamine-D-tryptophan and its preparation in pure form by crystallization and/or traditional chromatography on a column of silica gel.

The LEVEL of TECHNOLOGY

D-Isoglutamine-D-tryptophan (also known as H-D-iGlu-Trp-OH or thymodepressin [Thymodepressin]) is a synthetic generegulatory dipeptide created for the treatment of autoimmune diseases, including psoriasis (Sapuntsova, S. G., et al. (May 2002), Bulletin of Experimental Biology and Medicine, 133(5), 488-490). Thymodepressin considered Russia an effective tool for the treatment of psoriasis (U.S. patent 5736519), where the drug is currently available in the market as the disodium salt in the liquid preparation for injection and intranasal. He is an immunosuppressant and selectively inhibits the proliferation of bone marrow cells, thus causing suppression of the immune system.

Known solid form of the disodium salt of D-isoglutamine-D-tryptophan is an amorphous powder which is hygroscopic and very difficult from a technological point of view. With ructure disodium salt of thymodepressin described in Kashirin, D. M., et al. (2000), Pharmaceutical Chemistry Journal, 34(11), 619-622. Monosodium salt of D-isoglutamine-D-tryptophan identified by the Chemical Abstracts Service (CAS) and are listed in the registers CAS REGISTRYSMbut there is no publication concerning its preparation and physical properties. Powder or amorphous form of connection, such as D-isoglutamine-D-tryptophan is intended for pharmaceutical use may create operational problems due to problems with bulk density, hygroscopicity and varying water content, which cannot be adjusted by drying under vacuum. D-Isoglutamine-D-tryptophan is a dipeptide, and drying the amorphous form at elevated temperature, for example 80 to 100°C under vacuum is not recommended.

The only way of synthesis of H-D-iGlu-D-Trp-OH, well-known from the literature, disclosed in U.S. patent 5736519. In accordance with the method described in Example 1 of U.S. patent 5736519 depicted in this description as Scheme 1, Boc-D-Glu-OH (1.1) is reacted with 1,3-dicyclohexylcarbodiimide (DCC) with the formation of cyclic anhydride (1.2). After removal of dicyclohexylamine (DCM, DCU) is filtered, the anhydride (1.2) reacts with H-D-Trp-OH with the formation of a mixture of dipeptide Boc-D-iGlu-D-Trp-OH (1.3) and Boc-D-Glu-D-Trp-OH (1.4). The combined output of crude Boc-D-iGlu-D-Trp-OH (1.3) and Boc-D-Glu-D-Trp-OH (1.4) is 70%. However, the mixture contains less than 35% of the target intermediate with the unity of Boc-D-iGlu-D-Trp-OH (1.3). The protective group Boc is removed, mixing the solutions of (1.3) and (1.4) in formic acid as the solvent at 40°C for 1 hour. The relation (1.3) and (1.4) to formic acid is approximately 1 g:8 ml (wt./vol.). The product is a mixture of H-D-iGlu-D-Trp-OH (1.5) and H-D-Glu-D-Trp-OH (1.6). Because peptides (1.5) and (1.6) are in equal number, cleaning requires ion-exchange chromatography using pyridine acetate buffer. The yield of the target product H-D-iGlu-D-Trp-OH (1.5) is 35% of the number of Boc-D-iGlu-D-Trp-OH (1.3). Thus, the total yield of H-D-iGlu-D-Trp-OH (1.5) the number of Boc-D-Glu-OH is 12,25%.

Scheme 1: Synthesis of H-D-iGlu-D-Trp-OH in accordance with U.S. patent 5736519.

The method described in U.S. patent 5736519 has several disadvantages, as follows:

1. DC stage 1A can lead to the formation of other by-products, such as

About side products in the bonding of the peptides with DCC reported in Marder, O., and Albericio, F. (June 2003), Chemical Oggi (Chemistry Today), 6-32.

2. Removing protection with Boc-D-iGlu-D-Trp-OH (1.3) requires high temperature, and final purification of H-D-iGlu-D-Trp-OH requires extremely toxic solvent pyridine. Fever during unprotect (1.3) can lead to the formation of N-tert-butylaniline derivative (1.7) as an impurity (M. Löw, et. al. (1978), Hoppe-Seyler's Z. Physiol. Chem., 359(12):1643-51). In addition, the peptide may cilitates with the formation of glutarimide (1.8) (Pandit, U.K. (1989), Pure &Appl. Chem., Vol. 61, No. 3, pp. 423-426).

3. During the reaction of compounds formed only a mixture of Boc-D-iGlu-D-Trp-OH (1.3) and Boc-D-Glu-D-Trp-OH (1.4) in a 1:1 ratio. The maximum yield of (1.3) may not exceed 50% at the stage of compound 1B. A mixture of D-Glu-D-Trp-OH and D-iGlu-D-Trp-OH is formed at the end of the synthesis. The peptides must be separated by ion-exchange chromatography and preparative reversed-phase high-performance liquid chromatography (HPLC). The total yield of H-D-iGlu-D-Trp-OH (1.5) is 12,25%, and the purification preparative HPLC is a very long and inefficient process. Retention time for two such isomers, H-D-iGlu-D-Trp-OH (1.5) and H-D-Glu-D-Trp-OH (1.6), not shown. Repeated cycles of selection to increase the purity of the target isomer (1.5) are very inefficient. This method cannot be adapted to large scale production.

4. The opposite diastereoisomer L-isoglutamine-L-tryptophan (also known as H-L-iGlu-L-Trp-OH or Bestim [Bestim]) is an immunostimulant (see U.S. patent 5774452). Bestim used in the treatment of ulcers. It reduces inflammation in the stomach and the mucous membrane of the duodenum and causes regression of clinical symptoms and ulcer scarring (Tkacheva, A. et al. (2004), Eksp Klin Gastroenterol. (6):29-33, 163).

Synthesis monosodium salt (H-L-iGlu-L-Trp-OH (1:1) is shown in Scheme 2 (U.S. patent 5744452).

Scheme 2: Synthesis of [L-iGlu-L-Trp-O-]Na+in accordance with U.S. patent 5744452.

In the method of U.S. patent 5744452 in stage 1 as a by-product formed dicyclohexylamine, which must be removed by filtration. Stated that in the second stage triperoxonane acid removes the γ-o-benzyl ether fragment glutamic acid (2.2). Benzyl ether of (2.3) is removed by hydrogenation using ammonium formate as a hydrogen donor, palladium catalyst, sodium bicarbonate in isopropanol at elevated temperature to produce monosodium salt (H-L-iGlu-L-Trp-OH (2.4). Solid-phase synthesis of (2.4) is also described in the mentioned patent, but tryptophan fragment must be protected as formamide, and later, the protection must be removed. Other diastereomers, L-isoglutamine-D-tryptophan and D-isoglutamine-L-tryptophan, are also known compounds (U.S. patent 5916878).

Synthesis of H-D-iGlu-L-Trp-OH, and H-L-iGlu-D-Trp-OH is shown in Scheme 3 and Scheme 4, respectively (U.S. patent 5916878).

Scheme 3: Synthesis of H-D-isoglutamine-L-tryptophan in accordance with U.S. patent 5916878.

Scheme 4: Synthesis of H-L-isoglutamine-D-tryptophan in accordance with U.S. patent 5916878.

The methods described in Schemes 2, 3 and 4, can provide a synthesis regiospecific gamma-amide product (2.2), (3.2) and (4.2) without the formation of alpha-amide product, but they include a stage of hydrogenation during removal of the benzyl ether compounds (2.3), (3.2) and (4.2). This requires the use of large amounts of palladium catalyst. The second aspect relates to the partial recovery of the indole ring at the industrial scale. The third aspect concerns the education of glutarimide, 2-(3-amino-2,6-dioxo-piperidine-1-yl)-3-(1H-indol-3-yl)propionic acid, hydrogenation process. The fourth aspect relates to cost. The value of a derivative CBz-Glu-OBzl, such as (3.1) and (4.1), is almost twice the price of the corresponding Boc-Glu-OBzl in the production using technologies such as fine chemical synthesis. The ways in schemes 3 and 4 require purification of the final product by HPLC. The output value is 33% and 35.9 percent, respectively. Scheme 2 requires the use of triperoxonane acid, which introduces other impurities in the reaction. In addition, in the method of Scheme 2 using dicyclohexylcarbodiimide as a peptide condensing agent. Removal of trace impurities from this reagent is a serious problem in chemical production. Technology, thus, cannot be adapted to industrial produced the production and cannot be adapted for large-scale production of H-D-isoglutamine-D-tryptophan.

BRIEF description of the INVENTION

This invention relates to new stable crystalline form D-isoglutamine-D-tryptophan and allocation method of the compounds in pure form, free from inorganic salts by sedimentation from the water, without preparative reversed-phase high-performance liquid chromatography. The method is designed to obtain pure N-tert-butoxycarbonyl-D-isoglutamine-D-tryptophan and its diapir free from N-tert-butoxycarbonyl-D-glutamyl-D-tryptophan, and the conversion of N-tert-butoxycarbonyl-D-isoglutamine-D-tryptophan and its diapir in pure crystalline D-isoglutamine-D-tryptophan. New crystalline D-isoglutamine-D-tryptophan in this invention it is easy to clean. In comparison with the methods of the prior art described above, this invention offers several advantages as listed below.

First, D-isoglutamine-D-tryptophan get in crystalline form without the use of preparative high-performance liquid chromatography.

Second, a key intermediate compound Boc-D-iGlu-D-Trp-OH or salt of H-D-Glu-(γ-D-Trp-OMe)-α-OBzl HCl get high yield and high purity.

Thirdly, the method of conversion of Boc-D-iGlu-D-Trp-OH and its diapir or H-D-Glu-(γ-D-Trp-OMe)-α-OBzl HCl salt D-isoglutamine-D-tryptophan with high yield and high purity.

Fourth, the number of the traveler crystalline form D-isoglutamine-D-tryptophan in this invention is unknown from the previous prior art. It can be applied directly in the form of a liquid preparation with pH correction, which eliminates the need to use extremely hygroscopic and unstable disodium salt of D-isoglutamine-D-tryptophan.

This invention also relates to new stable ammonium salt of D-isoglutamine-D-tryptophan and its preparation of N-tert-butoxycarbonyl-D-isoglutamine-D-tryptophan, as well as the selection of such compounds in pure form by crystallization and/or traditional chromatography on a column of silica gel.

Monoamina Sol D-isoglutamine-D-tryptophan is a stable solid, suitable for dosing order to obtain preparative forms. Proposed schedule identifying part for identifying forms salts at different pH values.

A new method for producing D-isoglutamine-D-tryptophan and ammonium salts of D-isoglutamine-D-tryptophan (1:1) avoids the above-described production problems and makes possible the selection and processing of thymodepressin and monoammonium salt of thymodepressin traditional equipment for chemical processes.

The object of this invention is to propose a suitable method for the production of D-isoglutamine-D-tryptophan, which would give the substance of the medicinal product, completely free of other diastereomers, as obsujdalos the above, and the material's ability to maintain a stable form during a long storage period before the introduction of the drug.

Another object of this invention is to offer D-isoglutamine-D-tryptophan-free alpha-amide isomer D-glutamyl-D-tryptophan.

Another object of this invention is to propose a method of obtaining pure D-isoglutamine-D-tryptophan (H-D-iGlu-D-Trp-OH) of an acid additive salt of H-D-iGlu-D-Trp-OH, which gives the product are essentially or completely free from organic solvents and does not require purification by reversed-phase high-performance liquid chromatography. Solid D-isoglutamine-D-tryptophan is separated from the water.

Another object of this invention is to propose a method of obtaining pure D-isoglutamine-D-tryptophan (H-D-iGlu-D-Trp-OH) of the primary additive salt of H-D-iGlu-D-Trp-OH, which gives the product are essentially or completely free from organic solvents and does not require purification by reversed-phase high-performance liquid chromatography. Solid D-isoglutamine-D-tryptophan is separated from the water.

Another object of this invention is to propose a method, resulting in the formation of D-isoglutamine-D-tryptophan, essentially free from pollutants in the form of inorganic salts.

Another object of this invention is obtaining crystalline D-isoglutamine-D-is ryptophan with powder x-ray (XRPD), as shown in Fig. 1.

Another object of this invention is to provide monoammonium salt D-isoglutamine-D-tryptophan of the acid additive salts of D-isoglutamine-D-tryptophan, which gives the product are essentially or completely free from organic solvents and does not require purification by reversed-phase high-performance liquid chromatography. Solid ammonium salt of D-isoglutamine-D-tryptophan separated from the isopropanol and ammonia after treatment ion exchange resins to remove inorganic salts.

Another object of this invention is the production of monoammonium salt D-isoglutamine-D-tryptophan with XRPD shown in Fig. 2.

Another object of this invention is to obtain an amorphous monoammonium salt D-isoglutamine-D-tryptophan, which is essentially characterized by the IR spectrum with the Fourier-transformation (IR-AF), shown in Fig. 5.

Another object of this invention is to propose a method of obtaining monoammonium salt D-isoglutamine-D-tryptophan, essentially free from pollutants in the form of inorganic salts.

Another object of this invention is to propose a method of obtaining an acid additive salt, H-D-iGlu-D-Trp-OH, in particular the hydrochloride, of pure Boc-D-iGlu-D-Trp-OH.

Another object of this invention is to propose a method of obtaining pure dipeptide Boc-D-iGlu-D-Trp-OH no chromatogr the specific selection.

Another object of this invention is to provide a simple method of chromatographic allocation on a column of silica gel for purification of D-isoglutamine-D-tryptophan and its monoammonium salt.

Another object of this invention is to provide a schedule identifying the composition to determine the pH interval for selection D-isoglutamine-D-tryptophan and its a monovalent monocole. The dominant interval pH to precipitate D-isoglutamine-D-tryptophan of the water ranges from approximately 2.5 to approximately 3.0.

An acid additive salt of D-isoglutamine-D-tryptophan is obtained from the dipeptide Boc-D-iGlu-D-Trp-OH, the resulting ό hydrolysis of compounds of formula I:

,

where R1selected from the group consisting of C1-C4of alkyl and benzyl, and R2represents a C1-C4alkyl, provided that C4alkyl is tert-bootrom,

using a metal hydroxide in water and an inert solvent in the presence of methanol with the formation of Boc-D-iGlu-D-Trp-OH, free of other diastereomers. The hydroxide of a metal selected from the group consisting of lithium hydroxide, sodium hydroxide and potassium hydroxide.

The compound of the formula I, in turn, receive the peptide condensation of Boc-D-Glu(OH)-OR1and D-Trp OR2where R1and R2are such as identifying what about the above, with peptide condensing reagents such as HOBt and EDC. This method of synthesis of Boc-D-iGlu-D-Trp-OH provides significant advantages over previous prior art presented in U.S. patent 5736519, because the product is an exceptionally gamma peptide product Boc-D-iGlu-D-Trp-OH, and alpha-peptide product Boc-D-Glu-D-Trp-OH cannot be formed during the synthesis, because you are using Boc-D-Glu(OH)-OR1.

Removing protection from a pure dipeptide Boc-D-iGlu-D-Trp-OH with acid, such as chloromethane acid, triperoxonane acid, gives an acid additive salt. the pH of the acid-salt additive is from about 2.5 to about 3.0 obtaining thymodepressin in the form of a solid residue.

Alternative acid additive salt can be converted to the ammonium salt by treatment with aqueous material, ion exchange chromatography resin-based sulfonic acid. When removing the salt by elution using water ion-exchange resin is washed with a mixture of ammonia and isopropanol to obtain the crude ammonium salt, which is recrystallized from isopropanol and water to obtain pure monoammonium salt.

The solution is basically additive salts of D-isoglutamine-D-tryptophan is obtained by removing the protection with the acid, in particular to unprotect help the firm HCl, of the compounds of formula I, where each of R1and R2independently selected from the group consisting of C1-C4of alkyl and benzyl, with the receipt of an acid additive salt diapir H-D-Glu-(γ-D-Trp-OR2)-α-OR1which then is treated with a metal hydroxide in water and an inert solvent in the presence of methanol with the formation of the primary additive salt of H-D-iGlu-D-Trp-OH. The hydroxide of a metal selected from the group consisting of sodium hydroxide, lithium hydroxide and potassium hydroxide. The extraction solvent is not miscible with water, removes organic impurity in the organic phase; the aqueous fraction was separated and adjusted pH to values from approximately 6 to approximately 7 using a metal hydroxide. After evaporation of the solvent to reduce the amount of solvent to solute and solvent in less than about 1:8, where the dissolved substance is a peptide D-isoglutamine-D-tryptophan in the form of a basic additive salt, the pH of the resulting solution is basically additive salt is from about 2.5 to about 3.0 by using an inorganic acid to cause precipitation of D-isoglutamine-D-tryptophan.

Although does it limit in any way the context and practice of this invention by theory or practice of the invention and its possible and explanations it is believed that at a pH of from approximately 2.5 to approximately 3.0 schedule identification of the structure of Fig. 7 and Fig. 8 shows that the main forms of thymodepressin are free peptide (H-D-iGlu-D-Trp-OH) and monovalent salt. Since the solubility of H-D-iGlu-D-Trp-OH in water, not containing organic solvents, low (solubility <23 mg/ml), the compound precipitates from the solution in pure form. XRPD of the material shown in Fig. 1.

Manufacturing monoammonium salt solution thymodepressin at a pH of from about 6.0 to about 8.0 to cleanse ion exchange to remove salt. Regenerating solution on the basis of ammonia gives a clean monoammonium salt after crystallization from isopropanol and water. Although it can be assumed that monoammonium salt is unstable and can once again become free of the dipeptide, in practice, the connection actually is stable for more than two years. The applicant invented monoammonium salt thymodepressin, which is a new stable chemical object, which can easily crystallizing from isopropanol and water. Properties of the resulting crystalline material is shown in Fig. 2.

The above methods give a clean thymodepressin and monoammonium salt without reverse-phase HPLC on an industrial scale, but thymodepressin and m is nonammonia salt can be removed by conventional chromatography on a column of silica gel using the above conditions. Thus, instead of discarding portions of thymodepressin in the mother solution, crystallization optionally, the filtrate may be one stripped off and additionally purified by chromatography on a column of silica gel.

BRIEF DESCRIPTION of FIGURES

Crystalline salts of this invention are described in the Examples below.

Fig. 1 represents a graph XRPD of crystalline D-isoglutamine-D-tryptophan. Parameters XRPD can also be represented in the values of the interplanar distance d, Bragg angle 2θ and relative intensity (expressed as the percentage relative to the most intense line), as shown below.

3,3 2,444
Angle [°2θ]The value of d [Å]Relative intensity [%]
to 6.6713,2393
11,097,9754,4
11,777,5151,2
to 13.296,6554
14,266,20511,3
15,5833,3
the value of 16,815,26928,9
17,275,1330,4
18,354,83212,2
18,87the 4.795,8
20,05at 4,42463,6
20,94,24733,2
22,034,03217,1
22,88of 3.884100
23,743,74497,9
24,543,62541,9
25,443,49920,3
25,693,46512,1
26,313,38416,4
2727,4
27,753,21224,9
as opposed to 28.18 per3,16419,3
28,793,0996,8
29,133,0636,2
29,912,98579,2
31,042,8798,6
31,492,83933,7
32,542,7494,4
33,292,6899,3
33,972,63710,5
34,992,562the 17.3
35,542,52421,8
36,142,4835,1
36,745,9
37,352,4067,7
38,312,34825,6
39,012,30720,3

The sample powder obtained from a conventional technique front of the packing and analyzed the system diffractometer D8 Discovery source Cu-kα operating mode 45 kV/45 mA. The system is equipped with 2D proportional to the detector area (GADDS). Experimental data were obtained on two frames with contact 600 seconds for each, which covered the interval 3o-35o(2θ). The obtained 2D diffraction images were further integrated to obtain the standard, I vs. 2θ, the nature of diffraction. The data were processed using a variety of software for data processing Bruker AXS, including: Eva™ 8.0 and Topas™, version 2.1 (for the analysis of the approximation of the profile and, if necessary, scaling).

Fig. 2 is an XRPD crystalline monoammonium salt D-isoglutamine-D-tryptophan. Parameters XRPD can also be represented in terms of inter-planar distance d, Bragg angle 2θ and relative intensity (expressed as the percentage relative to h is the most intense line), as follows:

9,8
Angle [°2θ]The value of d [ Å]Relative intensity [%]
9,299,5174,1
12,197,2584,5
13,936,35476,2
15,175,83727,4
16,495,3719,8
17,185,1573
18,564,77831,6
18,884,69610,5
20,024,431100
22,283,9863
23,313,8144,6
23,663,757
24,033,752,9
24,373,64926,3
25,073,54911,4
25,613,4755,6
25,963,435
27,623,22729,7
28,123,1755,7
28,493,13112,2
29,523,02323,1
30,272,9513,7
30,642,9157,9
31,312,85411,8
31,72,82129,6
32,162,781 19,2
32,812,72816
33,782,6527,4
34,142,6255,1
35,762,50916
36,942,431the 11.6
37,582,39125,6
38,032,36412,5
to 39.222,2951,9

Spectra, powder x-ray D-isoglutamine-D-tryptophan and its ammonium salt are shown in Fig. 1 and Fig. 2 below. It should be understood that values of 2 theta for a chart of powdered x-rays may vary from one device to another or from one sample to another, that is, the values given should not be regarded as absolute.

Fig. 3 presents the XRPD of amorphous form D-isoglutamine-D-tryptophan.

Fig. 4 is a characteristic absorption spectrum in the infrared (IR) region of the crystal, monogamma the applications of salt D-isoglutamine-D-tryptophan.

Fig. 5 represents a characteristic absorption spectrum in the infrared (IR) region amorphous monoammonium salt D-isoglutamine-D-tryptophan.

Fig. 6 is a characteristic absorption spectrum in the infrared (IR) region of the crystalline D-isoglutamine-D-tryptophan.

Fig. 7 illustrates the calculation of the identification of the composition of the dipeptide H-D-iGlu-D-Trp-OH and its salts using the values of pKa for the acid and amine groups. LH2is a form of dibasic acid of the peptide H-D-iGlu-D-Trp-OH, LH is a salt of monocarboxylic acid H-D-iGlu-D-Trp-OH. An example of LH is monoammonium salt. L denotes the form of dibasic salt, and its example is the disodium salt of the peptide H-D-iGlu-D-Trp-OH.

Fig. 8 illustrates the calculation of the identification of the composition of the dipeptide H-D-iGlu-D-Trp-OH and its salts using experimentally derived values of pKa for the acid and amine groups. LH2is a form of dibasic acid of the peptide H-D-iGlu-D-Trp-OH, LH is a salt of monocarboxylic acid H-D-iGlu-D-Trp-OH. An example of LH is monoammonium salt. L denotes the form of dibasic salt, and its example is the disodium salt of the peptide H-D-iGlu-D-Trp-OH.

DETAILED description of the INVENTION

In this description, the term “Boc-D-Glu(OH)-OR1” denotes the structure:

if R1the submitted is a benzyl, from a chemical point of view, the connection is an alpha-benzyl ester 2-tert-butoxycarbonylamino-D-glutamic acid.

In this description, the term “D-Trp-OR2” denotes the structure:

if R2represents methyl, the compound is a methyl ester of D-tryptophan.

In this description, the term “Boc-D-Glu-(γ-D-Trp-OR2)-α-OR1” denotes the structure:

In this description, the term “H-D-Glu-(γ-D-Trp-OR2)-α-OR1” denotes the structure:

if R1represents benzyl, R2represents methyl, the compound is a

If R1represents methyl, R2represents methyl, the compound is a

In this description, the term “thymodepressin” means the dipeptide H-D-iGlu-D-Trp-OH with the chemical structure:

.

It can also be designated as H-D-Glu-(γ-D-Trp-OH)-OH.

An acid additive salt is a salt formed by the reaction of amine H-D-iGlu-D-Trp-OH with inorganic acids, including chloroethanol acid, sulfuric acid, Hydrobromic acid, phosphoric acid, etc. or organizes the mi acids, including formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, triperoxonane acid, benzoic acid, salicylic acid, benzosulfimide acid and toluensulfonate acid. It can also be formed by removing the protection derived from Boc-D-iGlu-D-Trp-OH with acid.

Basically additive salt is a salt formed by the reaction of carboxylic acid H-D-iGlu-D-Trp-OH with inorganic bases including sodium hydroxide, lithium hydroxide, potassium hydroxide, etc.

This invention is directed to a method of obtaining H-D-iGlu-D-Trp-OH and its ammonium salt, free from inorganic salts, an acid additive salt of H-D-iGlu-D-Trp-OH, which is preferably obtained from the dipeptide Boc-D-iGlu-D-Trp-OH. Boc-D-iGlu-D-Trp-OH is obtained from Boc-D-Glu(OH)-OR1and D-Trp OR2where R1selected from the group consisting of benzyl and C1-C4of alkyl, and R2represents a C1-C4alkyl, provided that C4alkyl represents tert-butyl.

This invention is also directed to a method of obtaining H-D-iGlu-D-Trp-OH from the solution basically additive salt of H-D-iGlu-D-Trp-OH, which preferably is are square of an acid additive salt of the dipeptide H-D-Glu-(γ-D-Trp-OR 2)-α-OR1where each of R1and R2independently selected from the group consisting of benzyl and C1-C4the alkyl.

PREFERRED OPTIONS

Within a few aspects of this invention, which are formulated in the brief description of the invention, the sequence of stages of the method and their relative preference described below.

In the embodiment, the present invention proposes a method of obtaining in the aqueous phase free from inorganic salts H-D-iGlu-D-Trp-OH, which includes:

(a) obtaining a solution of an acid additive salt of H-D-iGlu-D-Trp-OH in the aquatic environment, mainly containing organic solvent; or the receiving solution is basically additive salt of H-D-iGlu-D-Trp-OH in the aquatic environment, mainly containing organic solvent;

(b) correction of pH to pH corresponding to the predominant form for the dibasic form with a solution of a hydroxide of an alkali metal or inorganic acid to cause precipitation of H-D-iGlu-D-Trp-OH;

(c) isolation of the precipitated H-D-iGlu-D-Trp-OH; and

(d) vacuum drying the product obtained in stage (c), with the formation of H-D-iGlu-D-Trp-OH.

In another embodiment, the present invention features a crystalline form H-D-iGlu-D-Trp-OH, representing D-isoglutamine-D-tryptophan, which is characterized by XRPD, presented in the description of the figures.

In another embodiment, d is spent invention features a crystalline H-D-iGlu-D-Trp-OH, representing D-isoglutamine-D-tryptophan, which is characterized by XRPD, illustrated in Fig. 1.

In another embodiment, the present invention proposes a method of obtaining monoammonium salt of H-D-iGlu-D-Trp-OH, free from inorganic salts, where the method includes the following stages:

(a) obtaining a solution of an acid additive salt of H-D-iGlu-D-Trp-OH in the aquatic environment, mainly containing organic solvent;

(b) correction of pH to pH corresponding to the predominant form, to form monovalent salt with a solution of a metal hydroxide;

(c) treating the solution from step (b) ion exchange resin and elution with water for ion exchange of the metal salt in solution in hydrogen ion up until the pH will be from about 5.7 to about 7,0;

(d) contact ion-exchange resin with a regenerating solution on the basis of ammonia, which carries out the exchange of ions in solution on the target H-D-iGlu-D-Trp-OH, contained in the ion-exchange resin, with education, thus, the eluate regenerating solution containing the ammonium salt of H-D-iGlu-D-Trp-OH;

(e) evaporating the solvent from the solution from step (d) to obtain the crude ammonium salt;

(f) dissolving the ammonium salt from step (e) in the water and slow addition of isopropanol so that a precipitate of monoammonium salt; and

(g) vacuum drying is the product from step (f) with the formation of the crystalline form of the ammonium salt of H-D-iGlu-D-Trp-OH (1:1).

Alternative instead of stages (f) and (g) the method comprises the following stages:

(h) processing of material from step (d) by chromatography on silica gel using isopropanol and ammonia solution as eluent; and

(i) freeze drying the product from step (h) with the formation of the amorphous form of the ammonium salt of H-D-iGlu-D-Trp-OH (1:1).

In another embodiment, the present invention proposes a method of obtaining monoammonium salt of H-D-iGlu-D-Trp-OH crystalline H-D-iGlu-D-Trp-OH, free from inorganic salts, where the method includes the following stages:

(a) adding H-D-iGlu-D-Trp-OH for less than one equivalent of a solution of ammonium hydroxide;

(b) correction of pH to 6-7 with ammonium hydroxide;

(c) evaporating the solvent to form oil; adding isopropanol with stirring to cause precipitation of monoammonium salt;

(d) isolation of the precipitated ammonium salt of H-D-iGlu-D-Trp-OH and

(e) vacuum drying the product obtained in stage (c), with monoammonium salt of H-D-iGlu-D-Trp-OH.

In another embodiment, the present invention features a crystalline ammonium salt of H-D-iGlu-D-Trp-OH (1:1), which is characterized by XRPD, presented in the description of the figures.

In another embodiment, the present invention features a crystalline ammonium salt of H-D-iGlu-D-Trp-OH (1:1), which is characterized by XRPD shown in Fig. 2.

Another is ariante the present invention offers an amorphous ammonium salt of H-D-iGlu-D-Trp-OH (1:1), which is characterized by a spectrum of IR-FC (IR), shown in Fig. 5.

In another embodiment, the present invention proposes a method of obtaining an acid additive salt of D-isoglutamine-D-tryptophan, where salt is a H-D-iGlu-D-Trp-OH hydrochloride and where the method includes:

(i) basic hydrolysis of compounds of formula I:

,

where R1selected from the group consisting of C1-C4of alkyl and benzyl, and R2represents a C1-C4alkyl, provided that C4alkyl is tert-bootrom,

using a metal hydroxide in water and an inert solvent in the presence of methanol with the formation of Boc-D-iGlu-D-Trp-OH, free of other diastereomers;

(ii) removal using hydrogen chloride protection with Boc-D-iGlu-D-Trp-OH from the stage (i) in an inert organic solvent; and evaporating the solvent with the formation of the hydrochloride H-D-iGlu-D-Trp-OH.

In another embodiment, the present invention proposes a method of obtaining a solution of the acid-additive hydrochloride H-D-iGlu-D-Trp-OH, where the method includes:

(a) hydrogenation of the compounds of formula II:

,

where R1represents benzyl, and R2selected from the group consisting of benzyl, and hydrogen

using palladium on coal in methanol or ethanol;

(b) purification of crude H-D-iGlu-D-Trp-OH from stage (a) chromatog what afia on silica gel using isopropanol and water as eluent and

(c) processing the material from step (b) chloroethanol acid in water to form a solution of the hydrochloride H-D-iGlu-D-Trp-OH in water.

In the above-mentioned two ways of obtaining the acid additive salts of D-isoglutamine-D-tryptophan of the compounds of formula I is preferred in comparison with getting from the compounds of formula II from the point of view of chemical intermediates.

In another embodiment, the present invention proposes a method of obtaining a solution of a basic additive salt of H-D-iGlu-D-Trp-OH, where the method includes:

(a) removal using acid-protected dipeptide N-Boc-D-Glu-(γ-D-Trp-OR2)-α-OR1where each of R1and R2independently selected from the group consisting of C1-C4of alkyl and benzyl;

(b) basic hydrolysis of the product from step (a) with a metal hydroxide in water and an inert solvent in the presence of methanol, where the hydroxide of a metal selected from the group consisting of sodium hydroxide, potassium hydroxide and lithium hydroxide;

(c) extraction of the material from step (b) solvent not miscible with water, and separating the aqueous fraction;

(d) correction of the pH of the water fraction from step (c) to values in the range of from about 6 to about 7 and

(e) evaporating the solvent from the solution from step (d) to form a solution containing a ratio of about one is part of the dissolved substance at less than about 8 parts of water, where the dissolved substance is a basic additive salt of D-isoglutamine-D-tryptophan.

In another embodiment, the present invention proposes a new define derived H-D-Glu-(γ-D-Trp-OR2)-α-OR1hydrochloride, where each of R1and R2independently selected from the group consisting of benzyl and C1-C4the alkyl.

Compound H-D-Glu-(γ-D-Trp-OR2)-α-OR1unknown in the previous prior art. These compounds may be used as intermediate compounds to obtain the dipeptide D-isoglutamine-D-tryptophan. Alternative H-D-Glu-(γ-D-Trp-OR2)-α-OR1hydrochloride can be used in pharmaceutical drug where hydrolysis of the ester occurs in situ, to obtain the D-isoglutamine-D-tryptophan in the manufacture of the drug.

In the embodiment, the present invention proposes a schedule identifying the structure illustrated in Fig. 8, to highlight the H-D-iGlu-D-Trp-OH at a pH of from approximately 2.5 to approximately 3.0.

The WAY to OBTAIN

The present invention features, as shown below in Scheme 5, a reliable method of synthesis of N-(tert-butoxycarbonyl)-D-isoglutamine-D-tryptophan with high output, with the specified reliable method is missing in the previous prior art (for example, U.S. patent 5736519).

Scheme 5

In the method according to D. nomu the invention a solution of Boc-D-Glu(OH)-OR 1where R1represents benzyl, in an inert solvent is reacted with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), hydroxybenzotriazole (HOBt) and diisopropylethylamine (DIPEA). The preferred temperature is from about 5 to about -5°C, and the preferred solvent is dichloromethane. After stirring for from about 5 to about 30 minutes, preferably about 15 minutes, added dropwise a solution of salt of D-Trp-OR2HCl, where R2represents methyl, diisopropylethylamine (DIPEA). The resulting solution was stirred at ice temperature, preferably from about -5 to about 5°C for about 1 hour and then at room temperature for from about 12 to about 20 hours, preferably about 16 hours. The product Boc-D-iGlu-D-Trp-OR2)-α-OR1distinguish traditional means. The connection can be easily crystallized from ethyl acetate and hexane. Two synthetic impurities present in trace quantities connections (A) and (B), which can be removed by recrystallization. It is believed that both compounds are formed from the reagent HOBt.

Fluids Boc-D-Glu-(γ-D-Trp-OR2)-α-OR1in alcohol is mixed with a solution of sodium hydroxy is and. The preferred amount of sodium hydroxide is from about 2.5 to about 5 equivalents per equivalent of diapir. It is more preferable to use a molar ratio of from about 2 to about 3.5 mol of NaOH per mole defimage connection. The preferred ratio of the solvent is approximately 2 ml of alcohol per 1 ml of water, and the preferred ratio of NaOH and water is approximately 1 g per 20 ml of This method of isolation involves the extraction of the reaction mixture with ethyl acetate, and, therefore, any organic impurity is removed at this stage of the synthesis. After acidification of the aqueous fraction is extracted with an organic solvent such as ethyl acetate. Dibasic N-(tert-butoxycarbonyl)-D-isoglutamine-D-tryptophan (Boc-D-iGlu-D-Trp-OH) was isolated by traditional means in the form of solids. Isolated output for the two combined stages is 89%. This rate exceeded the methods of the prior art (U.S. patent 5736519). The new method according to this invention is additionally illustrated in the examples below.

Detailed research and monitoring stages of hydrolysis by the applicant have shown that the compound Boc-D-iGlu-D-Trp-OR2)-OR1where R1represents benzyl and R2represents methyl, first interacts with methanol with about the education Boc-D-iGlu-D-Trp-OMe)-OMe, after that connection is hydrolyzed to dibasic acids. The applicant determined that methanol is required for the rapid hydrolysis of alpha-benzyl ester. Due to the large volume of ethyl acetate required for extraction of the product, the applicant has invented a two-phase methodology for the efficient hydrolysis of Boc-D-iGlu-D-Trp-OR2)-OR1. Stirred mixture of Boc-D-iGlu-D-Trp-OR2)-OR1in tert-butylmethylether ether (MTBE) and a solution of a metal hydroxide, such as a solution of lithium hydroxide or sodium hydroxide. The preferred ratio of metal hydroxide and Boc-D-iGlu-D-Trp-OR2)-OR1is in the range from approximately 2.0 to 2.5 to 1. Add the methanol and the mixture is vigorously stirred for a period of from about 1 to about 6 hours, preferably from about 1.5 to about 2.5 hours. From a mixture secrete the organic phase by traditional means. This technique eliminates the need to use a large amount of ethyl acetate for extraction and illustrated in the examples below.

The traditional way to remove the protection from the compound Boc-D-iGlu-D-Trp-OH with organic acids with the formation of the dipeptide H-D-iGlu-D-Trp-OH, which requires serious treatment. There are numerous disadvantages of the methods of the prior art (U.S. patent 5736519)using formic acid, the ri 40°C to remove protection from a mixture of Boc-D-iGlu-D-Trp-OH and Boc-D-Glu-D-Trp-OH with the formation of a mixture of H-D-iGlu-D-Trp-OH and H-D-Glu-D-Trp-OH. Ion-exchange chromatography and reversed-phase HPLC is used to separate the product. The output is low, and the technique cannot be adapted to large scale production. Removing protection from N-tert-butoxycarbonyl group using triperoxonane acid or formic acid forms an ion tert-BUTYLCARBAMATE, which can react with indole nitrogen with the formation of N-tert-Putilkovo product (Löw, M., et. al. (1978), Hoppe-Seyler's Z. Physiol. Chem., 359(12):1643-51). Education glutarimide (1,8) (Pandit, U.K. (1989), Pure &Appl. Chem., Vol. 61, No. 3, pp. 423-426) is another problem.

The applicant determined that the acid additive salt according to this invention, in particular crude hydrochloride, can be easily obtained with HCl in an inert solvent, such as ethyl acetate, at a low temperature, preferably from about 0°C to ambient temperature. Evaporation of the solvent gives thymodepressin hydrochloride, which is used to obtain thymodepressin.

For a forecast of the required pH for precipitation of thymodepressin in the form of a dibasic acid H-D-iGlu-D-Trp-OH, by the applicant was a theoretical calculation on schedule identification of the composition, and it was concluded that thymodepressin exists in different form at pH values from approximately 2.5 to approximately 3.0. This pickup is allowed to invent the method of selection of thymodepressin without chromatography.

Fig. 7 illustrates this calculation. In Fig. 7 LH2is a form of dicarboxylic acid of the peptide H-D-iGlu-D-Trp-OH (i.e. thymodepressin), LH is a form salts of monocarboxylic acids of the peptide H-D-iGlu-D-Trp-OH; one such example is monoammonium salt, L represents the form of salts of dicarboxylic acids of the peptide H-D-iGlu-D-Trp-OH; one such example is a disodium salt, and LH3represents an acid additive salt of thymodepressin. The X-axis shows the pH of the solution. The Y axis shows % of education with respect to L (typical terminology software) and gives the molar fraction form, present at a particular value of pH. At a pH of from approximately 2.5 to approximately 3.0 the majority (80%) of the dipeptide exists as a form of dicarboxylic acid, and may be precipitated from a solution, if it is insoluble in water. Our study shows that the shape of the dicarboxylic acid obtained in this way shows that the solubility in water is approximately 23 mg/ml At a pH of approximately 7.0 and 100% of the forms submitted by the form of a monocarboxylic acid. If the counterion is a sodium, the form is thymodepressin monolatry.

In practice, if the solution of the hydrochloride of thymodepressin dissolved in water and the pH brought priblizitelen what to 3.0 with stirring, slowly, a solid substance that is filtered from the mixture. The material purity according to LC exceeds 97% and is within the category of pharmaceutical purity of the active pharmaceutical ingredient. The authors determined that this method is superior to prior art from the standpoint of production and allocation of net thymodepressin (H-D-iGlu-D-Trp-OH). There is no need for ion-exchange and reversed-phase chromatography of the drug on the column, since the only by-product is sodium chloride, soluble in water. The technique is a direct method of isolation and purification of thymodepressin. It provides a theoretical graph identify the composition and method outperforms the methods of the previous prior art, which require bulky cleanup.

The pKa value of the acid and amine groups H-D-iGlu-D-Trp-OH was determined experimentally. The graph structure of the dipeptide, built using experimentally derived values of pKa, shown in Fig. 8. In Fig. 8 LH2represents thymodepressin, LH is a salt of monocarboxylic acid, L represents a salt of dicarboxylic acid, and LH3represents an acid additive salt of thymodepressin. On the X-axis shows the values of pH of the solution. On the Y-axis shows % education regarding the structure for L (typical terminology software), and registered molar fraction forms present at a particular value of pH. Concentration of 0.5 M is used to show the equivalence of 1 g of thymodepressin in 6 ml of water for the purposes of allocation. This figure shows that approximately 75% of thymodepressin (LH2) exists in the form of a dicarboxylic acid at a pH of 2.7. For this reason, thymodepressin precipitates at pH 2.7 and can be filtered. The mother liquor may be one stripped off to obtain the second portion of thymodepressin. The graph confirms theoretical prediction is a form dibasic acid H-D-iGlu-D-Trp-OH predominates at a pH of from approximately 2.5 to approximately 3.0; specified interval represents the pH value to obtain a form dibasic acid from water in the form of sediment. Since only approximately 80% of the material will be deposited in pure form, the volume of the mother liquor should be reduced and subjected to a second round of precipitation at a pH of from approximately 2.5 to approximately 3.0, preferably at a pH of about 2.7.

Removing protection with Boc-H-D-iGlu-D-Trp-OH with triperoxonane acid in an inert solvent gives salt triperoxonane acid. The inert solvent is a dichloromethane, and usually use a mixture triperoxonane acid and dichloromethane (1:1). Evaporation of solvent gives an oil which is dried under vacuum to removed the I residual solvent. The oil is dispersed in water. When bringing the pH to approximately 3.0 sludge solids white after mixing for a period of from about 12 to about 16 hours.

Upon receipt of an acid additive salt is preferable to use HCl in an inert solvent to form the hydrochloride. Alternative salt triperoxonane acid can be formed using the above described method. The use of salt chloroethanol acid as an acid additive salt is preferred because the removal of protection with Boc-D-iGlu-D-Trp-OH is more effective when using HCl in an inert solvent, for example 3 M HCl in ethyl acetate. The duration of the reaction is considerably higher when using triperoxonane acid. In addition, salt triperoxonane acid D-iGlu-D-Trp-OH contains several synthetic impurities, which become D-iGlu-D-Trp-OH in the case of deposition at a pH of from approximately 2.5 to approximately 3.0 in the water. Impurities must be removed by repeated recrystallization.

Starting material Boc-D-iGlu-D-Trp-OH is obtained using methodology described earlier. An acid additive salt should be dried under vacuum, to ensure the absence of organic solvents and volatile impurities. In the case of deposition of thymodepressin of water recip is jut a solution of an acid additive salt in the water. The ratio of acid additive salt and water is in the range from about 1:5 to about 1:10. Even more preferably the ratio of the acid-salt additive is in the range from about 1:6 to about 1:8. A solution of a metal hydroxide, typically sodium hydroxide solution, is used for precipitation of the product, but can be used potassium hydroxide and the solutions of hydroxides of other metals.

Raw H-D-iGlu-D-Trp-OH can also be obtained by hydrogenation of compounds of formula II:

,

where R1represents benzyl, and R2selected from the group consisting of benzyl, and hydrogen using palladium on coal in methanol or ethanol. After filtration of the catalyst the filtrate is evaporated to a oil which is further purified by chromatography on silica gel using isopropanol and water as eluent. The obtained H-D-iGlu-D-Trp-OH can be converted to a salt of H-D-iGlu-D-Trp-OH hydrochloride in water using chloroethanol acid.

The solution is basically additive salts of D-isoglutamine-D-tryptophan is produced by removing the protection with the acid of the dipeptide Boc-D-Glu-(γ-D-Trp-OR2)-α-OR1where each of R1and R2independently selected from the group consisting of benzyl and C1-C4the alkyl. For example, the removal of protection with HCl with Boc-D-Glu-(γ-DTrp-OR 2)-α-OR1in an inert solvent, such as dichloromethane, gives Sol H-D-Glu-(γ-D-Trp-OR2)-α-OR1HCl. In the case of the combination, where R1represents benzyl and R2represents methyl, the product HCl.H-D-Glu-(γ-D-Trp-OR2)-α-OR1precipitated from dichloromethane and may be removed by filtration. Treatment of acid additive salts of a metal hydroxide in an inert solvent, such as methanol, with the aim of single-phase homogeneous hydrolysis or tert-butylmethylamine ether with the purpose of the two-phase hydrolysis gives basic additive salt of H-D-iGlu-D-Trp-OH in solution. When extraction of the reaction mixture with a solvent that is not miscible with water such as ethyl acetate or tert-butyl methyl ether, the aqueous fraction is neutralized to a pH from about 6 to about 7 and the solution is evaporated to reduce the volume to less than about 1 part solute to 8 parts water. The dissolved substance, as predicted by calculating the identify structure, as shown in Fig. 8 is a basic additive salt (in the form of monocarboxylate) H-D-iGlu-D-Trp-OH. If sodium hydroxide is used as the metal hydroxide, the permeate will be a monosodium form H-D-iGlu-D-Trp-OH in water. Correction of the pH of this solution to a range of approximately 2.5 to approximately 30 will cause precipitation of solid thymodepressin, H-D-iGlu-D-Trp-OH.

Monoammonium salt thymodepressin can be obtained directly from the dipeptide Boc-D-iGlu-D-Trp-OH. The crude acid additive salt, such as cleaners containing hydrochloride salt obtained in this way, as described above, is treated with ion exchange resins to remove inorganic salts. Thus, the solution of crude thymodepressin hydrochloride is dissolved in water and adjusted pH to values from about 6 to about 8. The solution is treated with ion exchange resins. Mainly resin is a resin-based sulfonic acid. An example is AMBERLYST® 15. Inorganic salt is removed by washing with water until pH of from about 5.7 to about 7. Ammonia is used as a regenerating means for producing ammonium salt of thymodepressin from the resin. Mostly use a concentrated solution of ammonia and isopropanol as regenerierung funds. The preferred ratio is a concentrated solution of ammonia and isopropanol in a ratio of from about 1 to about 3-4, with the ultimate proryvnym concentrated solution of ammonia and isopropanol in a ratio of 1 part of concentrated ammonia solution/1 part water/2 parts of isopropanol. The ammonia solution for washing is evaporated under reduced pressure to state oil that Chris is allsouth using isopropanol and water with the receiving monoammonium salt in a solid white color. The preferred ratio of isopropanol and water for recrystallization is in the range from about 5:1 to about 10:1. The need for column chromatography is missing.

In the previous prior art, there is no way to clean thymodepressin different from reversed-phase liquid chromatography of the drug. This method is extremely time consuming and expensive and may not be adapted to large scale production. The applicant determined that the raw thymodepressin can also be purified to pharmaceutical grade purity flash chromatography on silica gel using isopropanol and water as eluent. The preferred mobile phase is a mixture of isopropanol/water (from about 10:1 to about 5:1). The product distinguish traditional means.

In this way monoammonium salt can also be purified flash chromatography on silica gel using isopropanol and concentrated ammonia solution as eluent. The preferred mobile phase is a mixture of isopropanol/ammonia solution (from about 10:1 to about 5:1). The product distinguish traditional means.

Monoamina Sol D-isoglutamine-D-tryptophan obtained by crystallization using the m isopropanol and water, is a crystalline substance. On the other hand, if the solution monoammonium salt D-isoglutamine-D-tryptophan is dried by freeze drying, to obtain an amorphous material.

An extensive study was carried out to confirm that racemization chiral centers during purification on a column and the sequence of reactions is missing; the details shown in the examples below.

In accordance with this invention proposes a method of synthesis of Boc-D-iGlu-D-Trp-OH, free from alpha-isomer amide. We propose a method of conversion of Boc-D-iGlu-D-Trp-OH in an acid additive salt of thymodepressin, in particular the hydrochloride. Graph identification provides a deposition method of thymodepressin in pure form at a pH of about 3 in water. In addition, we offer a cleaning method of thymodepressin with a purity of less than 97% flash chromatography on a column using isopropanol and water as eluent. Another aspect of the present invention includes the traditional way obtain monoammonium salt of thymodepressin hydrochloride. Inorganic salt is removed by ion exchange resin, and monoammonium salt is mined using the regenerating solution is ammonia-based. Monoammonium salt can be obtained by crystallization in pure form. Also features a method of cleaning monoammonium salt more nyzkochastotnyy flash chromatography on a column of silica gel using isopropanol and water as eluent.

Additionally, the method of synthesis of thymodepressin unveiled in the previous prior art (U.S. patent 5736519), gives the raw thymodepressin, which must be purified by ion exchange chromatography and reversed-phase liquid chromatography of the drug. Department of Alfa-amide product H-D-Glu-D-Trp-OH from gamma-amide product of thymodepressin D-i-D-Glu-D-Trp-OH remains the most serious production problem. The process described in the previous prior art is unsuitable for large-scale production.

Application and introduction

The disodium salt of thymodepressin used for the treatment of psoriasis. Thus, crystalline thymodepressin and monoammonium salt thymodepressin according to this invention can be incorporated into pharmaceutical compositions for administration to a subject in a therapeutically active amount and in a biologically compatible form suitable for administration in vivo, i.e. in the form of peptides to the introduction, in which therapeutic effect superior to any toxic effects.

In accordance with the schedule identification of the structure as shown in Fig. 8, the predominant forms at neutral pH are monocarboxylate form of thymodepressin, that is, the monosodium salt of the dipeptide D-isoglutamine-D-tryptophan, if the counterion is a sodium. Dynatree the AI Sol D-isoglutamine-D-tryptophan is extremely hygroscopic and very difficult from the standpoint of processing and producing dosage forms. Crystalline thymodepressin according to this invention has an XRPD as shown in detail in Fig. 1, and its solubility in water is approximately 20 mg/ml Compound is the ideal candidate to replace the disodium salt in the manufacture of various drugs. Although the pH of the solution D-isoglutamine-D-tryptophan is approximately 3, can be carried out correction of the pH value with sodium hydroxide solution, sodium carbonate or sodium bicarbonate to a pH of from about 7 to about 7.4 for. Monoammonium salt according to this invention exists in crystalline and in amorphous form. Both forms of monoammonium salts have an extremely high solubility in water. Thus, they are also suitable candidates for recipes.

The introduction of a new crystalline thymodepressin and/or its monoammonium salt, as described herein, may be implemented with any of the conventional routes of administration of therapeutic drugs with systemic activity. These methods include dosage forms for oral, parenteral and other types of systemic or local application or application in the form of an aerosol.

Depending on the intended method of administration of the composition for application can exist in the form of a solid, tverdykh or liquid dosage forms, such as tablets, suppositories, pills, capsules, powders, liquids, sprays, suspensions and the like, preferably in dosage forms suitable for single introduction accurate doses. The composition will contain at least one conventional pharmaceutical carrier or excipient and crystalline thymodepressin or its pharmaceutically acceptable monoammonium salt, and may additionally include other medicinal agents, pharmaceutical agents, carriers, adjuvants, etc.

For solid compositions can be used in conventional non-toxic solid carriers include, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharinate, talc, cellulose, glucose, sucrose, magnesium carbonate and the like, pharmaceutical grade. The active compound, as defined above, can be entered into a suppository using, for example, polyalkylene glycols, such as propylene glycol, as the carrier. Suitable for pharmaceutical injection liquid compositions can, for example, be obtained by dissolving, dispersing, etc. an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as water, saline, aqueous dextrose, glycerol, ethanol and the like, with the formation of such act shall obom solution or suspension. Optionally, the pharmaceutical composition for injection may also contain minor amounts of nontoxic auxiliary substances, such as moisturizing or emulsifying means, the buffer means and the like, for example sodium acetate, sorbitan monolaurate, sodium, triethanolamine acetate, triethanolamine the oleate, etc. the Actual methods for such dosage forms are known or will be apparent to a person skilled in the industry; for example, see Remington: The Science and Practice of Pharmacy, David B. Troy (Ed.), Lipincott Williams & Wilkins, Philadelphia, PA, 21st Edition, 2006. The composition or formulation for administration in any case will contain the number of active(s) connection(s) in an amount effective to alleviate symptoms of the subject to be treated.

Parenteral administration generally represented by injection, subcutaneous, intramuscular or intravenous. The injection means can be made in traditional forms, such as liquid solutions or suspensions, solid forms suitable for the manufacture of a solution or suspension in liquid prior to injection, or as emulsions. Suitable auxiliary substances, such as water, saline, dextrose, glycerol, ethanol and the like, in Addition, optionally, the pharmaceutical composition for injection may also contain minor amounts of nontoxic auxiliary is a recreational substances, such as moisturizing or emulsifying means, the buffer means and the like, such as sodium acetate, sorbitan monolaurate, the triethanolamine oleate, etc.

For thymodepressin or monoammonium salt preferably oral or nasal (intrabronchial) introduction depending on the nature of the disorder to be treated.

For oral administration of pharmaceutically acceptable non-toxic composition was prepared by combining any of the commonly used excipients, pharmaceutical grade, such as mannitol, lactose, starch, magnesium stearate, sodium saccharinate, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. Such compositions take the form of solutions, suspensions, tablets, pills, capsules, powders, prolonged release formulations and the like, Such compositions can contain from approximately 1% to approximately 95% active ingredient, preferably from about 25% to about 70%.

Oral and nasal introduction into the lungs can also be carried out using aerosol dosage forms. For aerosol injection active ingredient is preferably supplied in finely ground form, together with the surface-active agent and a propellant. Typical percentages of the active ingredients is from approx the positive 0.01 to about 20 wt.%, preferably from about 0.04% to approximately 1.0%.

Surfactants usually must be non-toxic and preferably soluble in the propellant. A typical example of such tools are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as Caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, alasaarela and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride such as ethylene glycol, glycerin, erythritol, arabitol, mannitol, sorbitol anhydride hexitol derived from sorbitol (esters sorbitan present on the market under the name SPAN®), and polyoxyethylene and polyoxypropylene derivatives of these esters. Can be used mixed ethers, such as mixed or natural glycerides. Preferred surface-active agents are the oleates or sorbitan, for example, present on the market under the name ARLACEL®C (sorbitan sesquioleate), SPAN®80 (sorbitan monooleate) and SPAN®85 (sorbitan trioleate). The content of the surfactant may range from about 0.1% to about 20 wt.% of the composition, preferably from about 0.25% to about 5%.

The rest of the song is usually a propellant. See the military propellants are normally gaseous at ambient conditions and are condensed under pressure. Among suitable liquefied propellants - lower alkanes containing up to five carbon atoms, such as butane and propane, and preferably fluorinated or ferroresonance alkanes, such as those present on the market under the name FREON®. A mixture of the above components can also be used.

In the production of the aerosol container equipped with a suitable valve, fill in the appropriate propellant containing finely ground active ingredient and a surfactant. Thus, the ingredients contained at high pressure to release upon actuation of the valve.

For local administration, such compositions contain an effective amount of compounds of this class in a mixture of at least one pharmaceutically acceptable non-toxic carrier. Suitable spacing of the composition is from about 0.1% to about 10% active ingredient, and the rest are carriers, preferably from about 1% to about 2% active ingredient. The concentration of active ingredient in pharmaceutical compositions suitable for topical application, will vary depending on the specific activity of the compound, which is used depending on the present condition and subject to be treated. Suitable is ositelu or solvents medicines for local use of these compounds include creams, ointments, lotions, emulsions, solutions, etc.

For example, suitable ointment for topical application of the compounds according to this invention contains from about 15 to about 45% of saturated fatty alcohol containing 16 to 24 carbon atoms, such as cetyl alcohol, stearyl alcohol, beganovic alcohol and the like, and from about 45 to about 85 wt.% glycol solvent such as propylene glycol, polyethylene glycol, dipropyleneglycol and mixtures of these components. The ointment may also contain from about 0 to about 15 wt.% plasticizer such as polyethylene glycol, 1,2,6-hexanetriol, sorbitol, glycerol and the like; from about 0 to about 15 wt.%, binders, such as saturated fatty acids containing from 16 to 24 carbon atoms, for example stearic acid, palmitic acid, Bekenova acid, amides of fatty acids, for example, oleamide, palmitate, stearate, beginnig, and esters of fatty acids containing 16 to 24 carbon atoms, such as sorbitol monostearate, polyethylene-glycol monostearate, polypropylenglycol or appropriate monoether other fatty acids, such as oleic acid and palmitic acid; and from about 0 to about 20 wt.% tools that enhance penetration, such as dimethyl sulfoxide or dimethylacetamide.

Therapeutically active amount of a crystalline thymodepressin or its ammonium salt may vary in accordance with such factors as the pathological condition, age, sex and body weight of the individual. The treatment regimen may be adjusted to provide the optimum therapeutic response. In the whole scheme of daily introduction should include a dose in the range of from about 1 to about 200 mg of the peptide.

The following are examples of typical formulations, which in no way limit the context of the preparation of various pharmaceutical compositions:

IngredientsQuantity per tablet (mg)
The active ingredient25
Lactose, spray dried20
Corn starch153
Magnesium stearate2

The above ingredients are thoroughly mixed and pressed into tablets with a single line.

IngredientsThe number n is the tablet (mg)
The active ingredient100
Lactose, spray dried148
Magnesium stearate2

The above ingredients are mixed and introduced into hard gelatin capsule.

IngredientsQuantity per tablet (mg)
The active ingredient200
Lactose145
Corn starch50
Magnesium stearate5

The above ingredients are thoroughly mixed and pressed into tablets with a single line.

IngredientsQuantity per tablet (mg)
The active ingredient108
Lactose15
Corn starch25
Magnesium stearate2

The above ingredients are mixed and introduced into hard gelatin capsule.

IngredientsQuantity per tablet (mg)
The active ingredient150
Lactose92

The above ingredients are mixed and introduced into hard gelatin capsule.

Injectable, buffered to pH around 7, get with the following structure:

Ingredients
The active ingredient0.2 g
KH2PO4
KOH (1 n solution)
2 ml
the required amount to pH 7
Water (distilled, sterile)the required amount to 20 ml

Injectable, buffered to pH around 7, get with the following structure:

tr>
Ingredients
The active ingredient0.01 g
Water (distilled, sterile)required quantity up to 1 ml
NaOH (0,2 ad)the required amount to pH 7

Suspension for oral use are the following composition:

Ingredients
The active ingredient0.1 g
Fumaric acid0.5 g
Methylparaben2.0 g
Sugar granulation0.1 g
Sorbitol (70% solution)25,5 g
VEEGUM® K (Vanderbilt Co.)is 12.85 g
Flavor1.0 g
Dyesa 0.035 ml
Distilled waterthe required amount to 100 ml

Preparation for local p is imeneniya:

IngredientsGrams
Active connection0,2-2
SPAN® 602
TWEEN® 602
Mineral oil5
Vaseline10
Methylparaben0,15
Propylparaben0,05
BHA (bottled
hydroxyanisol)
0,01
Distilled waterthe required amount to 100 ml

All of the above ingredients, except water, are combined and heated to approximately 45°C with stirring. Then add sufficient amount of water with a temperature of approximately 45°C with vigorous stirring for emulsification of the ingredients and then add the required amount of water to 100 g

Below the invention is explained in detail with reference to Examples, but the invention is not limited to any education is om.

Example 1

Obtain N-α-tert-butoxycarbonyl-γ-D-glutamyl(α-benzyl ester)-D-tryptophan methyl ester or (2R)-tert-butoxycarbonylamino-(4R)-[2-(1H-indol-3-yl)-1-methoxycarbonyl-ethylcarboxyl]butyric acid benzyl ester or N-tert-Boc-D-Glu-(γ-D-Trp-OMe)-α-OBzl.

Method 1A:

Obtaining pure reference sample N-tert-Boc-D-Glu-(γ-D-Trp-OMe)-α-OBzl with purification by chromatography on silica gel.

With stirring to a cooled on ice to a solution of Boc-D-Glu-OBzl (6,00 g, 17.8 mmol) in CH2Cl2(70 ml) was successively added EDC (5,11 g of 26.6 mmol), HOBt (of 3.60 g of 26.6 mmol) and DIPEA (4,60 ml of 26.6 mmol). Then added dropwise a solution of H-D-Trp-OMe.HCl (6,77 g of 26.6 mmol) and DIPEA (4,60 ml of 26.6 mmol) in CH2Cl2(50 ml). The resulting mixture was stirred at ice temperature (-3°C to 0°C) for 1 hour, then allow to warm to room temperature and stirred for 16 hours. The reaction mixture is evaporated until dry. The residue is partitioned between EtOAc and a saturated solution of NaHCO3. The organic fraction is collected, washed with 10% citric acid solution and then with saturated saline. The organic fraction is dried over sodium sulfate, filtered and evaporated to a thick oil. The residue is purified column chromatography on silica gel using a gradient solvent mixture hexane/EtOAc (value 82, 7:3 and 3:7, vol/about.) as eluent to obtain specified in the header of the product (9,40 g, 98%) as a solid white color.1H NMR (DMSO-d6) δ ppm 10,86 (s, 1H), 8,31 (d, J=7,4 Hz, 1H), 7,49 (d, J=7.7 Hz, 1H), 7,31-7,35 (m, 7H), 7,14 (d, J=2.0 Hz, 1H), 7,06 (t, J=7.9 Hz, 1H), 6,98 (t, J=6,8 Hz, 1H), 5,12 (kV, J=5,9 Hz, 2H), 4,5 (kV, J=6,5 Hz, 1H), 3.96 points-a 4.03 (m, 1H), 3,55 (s, 3H), 2,98-and 3.16 (m, 2H), 2,18-of 2.24 (m, 2H), 1,86-of 1.95 (m, 1H), 1,71 and 1.80 (m, 1H), of 1.37 (s, 9H);13C NMR (DMSO-d6) δ ppm: 172,4 (C), 172,3 (C), 171,4 (C), 155,6 (C), 136,1 (C), 136,0 (C), 128,4 (CH), to 127.9 (CH), of 127.7 (CH), 127,1 (C), 123,6 (CH), 120,9 (CH), 118,4 (CH), to 117.9 (CH), byr111.4 (CH), 109,5 (C), 78,2 (C), 65,8 (CH2), 53,3 (CH), 53,16 (CH), of 51.7 (CH3), 31,3 (CH2), 28,2 (CH3), 27,1 (CH2), 26,4 (CH2); MS (ratio of mass and charge) 538 [M+1]+; analytical calculated for C29H35N3O7.0,5H2O: C, 63,72; H, only 6.64; N, of 7.69; found: C, 63,79; H, the 6.06; N, 7,65.

Method 1B:

Obtaining a pure sample of N-tert-Boc-D-Glu-(γ-D-Trp-OMe)-α-OBzl by recrystallization.

A suspension of Boc-D-Glu-OBzl (60,26 g, 178,6 mmol) in CH2Cl2(335 ml) is cooled to approximately -1oC and stirred for 15 minutes Then sequentially add DIPEA (46,70 ml, 268,0 mmol), HOBt (36,20 g, 268,0 mmol), EDC (51,38 g, 268,0 mmol). Then added dropwise a solution of H-D-Trp-OMe.HCl (68,25 g, 268,0 mmol) and DIPEA (46,70 ml, 268,0 mmol) in CH2Cl2(187 ml). The resulting mixture was stirred at ice temperature (-1°C to -5°C) for 2 hours, then allow to warm to room temp the atmospheric temperature and stirred overnight under nitrogen atmosphere.

The reaction mixture is evaporated until dry. The residue is partitioned between EtOAc (200 ml), a saturated solution of Na2CO3(100 ml) and H2O (150 ml). The aqueous fraction is again extracted with ethyl acetate (200 ml). The organic fraction is collected, washed with H2O (100 ml), 10% citric acid solution (2×200 ml) and saturated brine (60 ml). The organic fraction is dried over sodium sulfate, filtered and evaporated until dry. The residue is dissolved in ethyl acetate (141 ml), then add hexane (106 ml). The resulting suspension is stirred for 6 hours and filtered. The solid is thoroughly washed with hexane (100 ml), then dried under vacuum in an oven at 40°C, soaking at this temperature over night. Get the solid is nearly white (81,67 g, 85%). Data1H NMR confirmed the structure (see Example 1, Method 1A).

Method of 1C:

Obtaining a pure sample of N-tert-Boc-D-Glu-(γ-D-Trp-OMe)-α-OBzl without chromatographic purification and determination of synthetic impurities.

Boc-D-Glu-OBzl (48,0 g, 142,2 mmol) is dissolved in 270 ml of dichloromethane, and then cooled to 0-5°C with an ice bath. Added HOBt (23,8 g, 156,4 mmol) followed by addition of DIPEA (27,0 ml, 156, 4mm mmol) and stirred for 10 minutes EDC (38 g, 199,1 mmol) and pre-mixed solution of H-D-Trp-OMe (prepared from H-D-Trp-OMe.HCl [39,7 g, 156,4 mmol] and DI the EA [27,0 ml, 156,4 mmol] in 150 ml of dichloromethane is stirred at room temperature for 20 min successively added to the solution. The reaction mixture was stirred at 0°C, soaking at this temperature for 2 hours and then overnight at room temperature. The reaction mixture was poured onto 250 ml of distilled water and shaken out. The organic fraction was washed with 250 ml of 10% citric acid solution, 2×5% solution of NaHCO3and saturated salt solution. The organic fraction is dried over sodium sulfate and evaporated under vacuum to obtain foamy solid pale yellow color.

The solid is dissolved in about 250 ml of ethyl acetate and evaporated until dry. The operation is carried out twice with the formation of a waxy solid. The solid material was added 100 ml of ethyl acetate and stirred at room temperature. The mixture is stirred with moderate or high speed to the formation of the slurry with the consistency of the batter - this process takes approximately 45 minutes (stirring for a long period of time can lead to hardening of the solution in the form gelatinases substance). Then add 75 ml of hexane and the mixture is stirred for 10 minutes At this point add another 20 ml of ethyl acetate and the suspension was filtered immediately with the floor is the group of fluffy solid pale pink color. The solid is immediately washed with three times 30 ml of hexane, which helps to remove the pinkish coloration. The filtrate is collected and allow to stand still for 40 minutes Granular solid is precipitated from the filtrate. The mixture is filtered and the solid is washed three times with 10 ml of hexane.

The filtrate is collected and evaporated to a solid state. The solid is dissolved in 20 ml of ethyl acetate and stirred until the formation of the slurry with the consistency of the batter. Then add 40 ml of hexane and the mixture is stirred for 5 minutes the Mixture is filtered and the collected solid washed with hexane. The combined solids are dried overnight in an oven (35°C) under vacuum to constant weight. So, get 59.0 g (77.2 percent) specified in the connection header. TPL: 83,1 to 87.5°C. Data1H NMR identical to that described in Example 1, Method 1A. Purity according to HPLC (% peak area): 97,2%; retention time: 7,56 min; HPLC: column Waters Symmetry C 18, of 3.9×150 mm, 5 μm; mobile phase: 0.035% of HClO4pH 2/CH3CN gradient (min-% CH3CN) 0-35, 10-90, 12-90; flow rate: 1 ml/min; λ: 230, 260, 280 nm.

Analysis of impurities in the mother solution.

The results of the analysis of the mother liquor by TLC (mixture of ethyl acetate/hexane 50:50) shown in the table below. Both spots a and B fluoresce under UV light, but give a negative result ning grinovich tests. Spots product and the starting line give positive ninhydrin test. Samples a and B allocate column chromatography on silica gel, and their structure is clarified according to the1H NMR and MS/MS. The structure of a and B is shown below.

The value of Rf in a mixture of ethyl acetate/hexane 50:50:

SpotThe Rf-value
And0,80
B0,60
Product0,39

Spots a and B meet the impurities associated with HOBt. Impurities can be removed by recrystallization. Any trace impurities can be removed in the next stage of hydrolysis.

Example 2

Obtain N-α-tert-butoxycarbonyl-D-isoglutamine-D-tryptophan, or (2R)-tert-butoxycarbonylamino-(4R)-[1-carboxy-2-(1H-indol-3-yl)-ethylcarboxyl]-butyric acid or N-tert-α-Boc-D-iGlu-D-Trp-OH.

Method 2A:

Single-phase hydrolysis using NaOH.

With stirring to a solution of N-tert-Boc-D-Glu-(γ-D-Trp-OMe)-α-OBzl (3.7 g, 6,9 mmol) from Example 1, Method 1A, in methanol (40 ml) add a solution of NaOH (1.0 g, 25 mmol) in H2O (20 ml). The resulting solution was stirred at room temperature overnight. The reactions is nnow mixture was poured into 1 N. NaOH solution (100 ml) and the aqueous mixture washed with ethyl acetate (2×100 ml). The aqueous fraction is acidified with 3 N. HCl solution, then extracted with ethyl acetate (2×50 ml). The organic fractions combined, dried over sodium sulfate and evaporated until dry under reduced pressure. Get a solid white color (2.7 g, 91%). TPL: 148-158°C;1H NMR (DMSO-d6) δ ppm: 12,47 (W, 2H), was 10.82 (s, 1H), 8,21 (d, J=7.8 Hz, 1H), 7,53 (d, J=7.8 Hz, 1H), 7,34 (d, J=8,1 Hz, 1H), 7,13 (d, J=2.0 Hz, 1H), 7,06 (t, J=7.5 Hz, 2H), 6,98 (t, J=7,4 Hz, 1H), 4,46 (kV, J=5.3 Hz, 1H), 3,88-a 3.83 (m, 1H), 3,17-of 2.97 (DD, J=5,2 and 8.4 Hz, 2H), 2.23 to-2,10 (m, 2H), 1,90-to 1.82 (m, 1H), 1,75 by 1.68 (m, 1H), to 1.38 (s, 9H);13C NMR (DMSO-d6) δ ppm: 173,9 (C), 173,4 (C), which is 171,5 (C), 155,6 (C), 136,1 (C), 127,2 (C), to 123.5 (CH), 120,9 (CH), 118,4 (CH), 118,2 (CH), byr111.4 (CH), 109,9 (C)78,0 (C)53,1 (CH), 52,9 (CH), 31,7 (CH2), 28,2 (CH3), 27,2 (CH2), 26,7 (CH2); IR AF (KBr) ν: 3415, 3338, 2986, 1719, 1686, 1654, 1534, 1424, 1366, 1252, 1169, 1069, 744, 634, 429 cm-1; MS (ratio of mass and charge) 434 [M+1]+.

Method 2B:

Single-phase hydrolysis using LiOH.

With stirring to a chilled on ice (0°C to 5°C) solution of Boc-D-Glu-(γ-D-Trp-OCH3)-α-OBzl (of 46.06 g, 85,68 mmol) in methanol (200 ml) is added a solution of LiOH (10,78 g, 257,0 mmol) in H2O (136 ml). The resulting solution was stirred and kept at a temperature from 0°C to 10°C, soaking at this temperature for 3 hours. The reaction mixture was poured into a saturated solution of Na2CO3(100 ml) and H2O (150 ml), aq the second mixture is washed with ethyl acetate (2×150 ml). The aqueous fraction is acidified to pH 2-3 with 3 n HCl solution, then extracted with ethyl acetate (2×200 ml). The organic fractions combined, dried over sodium sulfate and evaporated until dry under reduced pressure. Get a solid white color (36,65 g, 98.7 per cent). Data1H NMR and MS/MS to confirm the structure (see Example 2, Method 2A).

Method 2C:

Obtain Boc-D-iGlu-D-Trp-OH without chromatographic purification using a two-phase hydrolysis process.

Lithium hydroxide (4.1 g, is 97.7 mmol) is dissolved in 35 ml of distilled water. Then add 65 ml of methyl tert-butyl ether (MTBE) followed by the addition of the dipeptide Boc-D-Glu-(γ-D-Trp-OCH3)-α-OBzl (25 g, 46.5 mmol), obtained as described in Example 1. Immediately formed a very thick suspension, and 15 ml of methanol and 15 MTBE added under vigorous stirring. Add another 2 ml of methanol and the solid slowly dissolved within about 5 minutes Immediately after the dissolution of the material solution becomes yellow/green color, while the upper organic fraction becomes pale green color, and the water fraction - yellow color. The reaction mixture was vigorously stirred at room temperature for 80 min; at this point of time in the organic fraction remains the starting material and the aqueous fraction contains the product contacts the ol by TLC: a mixture of EtOAC/hexane [1:1], about./vol.). The solution was poured into a separating the lake and 2 of the faction divide. The organic fraction was washed with 15 ml of water. The organic fraction becomes pink in the washing water. The combined aqueous fraction is washed twice with 30 ml of ethyl acetate. The aqueous fraction is acidified to approximately pH 2 by the addition dropwise of 16.6 ml of 6N chloroethanol acid at room temperature. The aqueous fraction is extracted with twice 50 ml of ethyl acetate. The maximum amount of methanol added during the second extraction to improve the solubility of the product in the organic fraction. The combined organic fractions are dried over sodium sulfate and evaporated in vacuo from the liquid yellow with obtaining a solid white color. The solid is dried overnight in an oven (28°C) under vacuum to constant weight.

So get to 18.6 g (yield 92%) specified in the connection header. TPL: 179,0-184,6°C; Data1H NMR was identical to that described in Example 2A; purity according to HPLC (% peak area): 98,3%; retention time: 5,33 min; HPLC: column Waters Symmetry C 18, of 3.9×150 mm, 5 μm; mobile phase: 0.035% of HClO4pH 2/CH3CN, gradient (T [min-% CH3CN) 0-20, 10-90, 12-90; flow rate: 1 ml/min; λ:230, 260, 280 nm.

Control of the above-mentioned reaction can be carried out using HPLC. In the Method 2C, above, the presence of benzyl JV the mouth, the resulting hydrolysis fragment benzyl ester, can be monitored using TLC (mixture of EtOAC/hexane [1:1, vol/about.] as eluent). Benzyl alcohol formed in the hydrolysis fragment benzyl ether. The impurity corresponding to the first spot is the same as in Example 1, Method 1C. Control using HPLC and analysis by LC/MS shows that Boc-D-Glu-(γ-D-Trp-OCH3)-α-OBzl first reacts with the base and methanol with the formation of Boc-D-Glu-(γ-D-Trp-OCH3)-α-OCH3, which is then rapidly hydrolyzed to obtain dibasic acid, Boc-D-Glu-(γ-D-Trp-OH)-OH or Boc-D-iGlu-D-Trp-OH.

Example 3:

Getting D-isoglutamine-D-tryptophan.

Method 3A:

Getting D-isoglutamine-D-tryptophan and its purification by recrystallization.

Boc-D-iGlu-D-Trp-OH (20,0 g, 46,14 mmol, from Example 2) is placed in a 3-necked round bottom flask with a volume of 1 l, equipped with a magnetic stirrer. Add ethyl acetate (300 ml) and the resulting suspension is cooled to -10°C in an ice bath. Gaseous HCl is bubbled through the cold suspension. The temperature interval from -4°C to -10°C is maintained during the reaction and the progress of the reaction monitored by HPLC. At some point in the heterogeneous reaction mixture becomes transparent homogeneous solution light pink color. After the conversion of starting material the reaction mixture was again Pribram who is in suspension. Volatile materials are then removed in vacuum to obtain solid light pink color. The solid is dissolved in 60 ml of demineralized water and the resulting solution was washed with dichloromethane (2×25 ml). Then the pH of the aqueous solution was adjusted approximately to 3.0 by addition of NaOH (10 M, approximately 3.6 ml) under cooling. The resulting solution is filtered to remove any residual particles of a solid substance. The filtrate is collected and vigorously stirred. The solid is separated by filtration. The filtrate is left for later use. Then the solid is again placed in a round bottom flask and add 30 ml of demineralized water. The mixture is vigorously stirred and the solid is separated by filtration. The filtrate is left for later use. The solid is then washed with ice-cold demineralized water (4×15 ml). The third wash water solution does not contain chlorides, which is confirmed by the negative results of trials using AgNO3(4% solution). The solid is air-dried, then placed in a vacuum oven at 36°C, soaking at this temperature over night to obtain 8.5 g (purity according to HPLC, % peak area: 98,3%).

The filtrates from the above stages are combined and carried out the same procedure recrystallization from gaining the m further 3.2 g of the product (purity according to HPLC, % peak area: 98,7%). The combined output for 2 servings is 11.7 g (75%).

Additional processing of the final filtrate gives a third portion (1.0 g, purity according to HPLC, % peak area: 82,0%).

1H NMR (D2O-NaOD, pH 7.0) δ ppm: to 7.64 (d, J=7.9 Hz, 1H), 7,43 (d, J=8,1 Hz, 1H), 7,19-7,16 (m, 2H), 7,10 (t, J=7,4 Hz, 1H), to 4.52-4,48 (m, 1H), 3,48 (t, J=6,1 Hz, 1H), 3,34 be 3.29 (m, 1H), is 3.08-to 3.02 (m, 1H), 2,30-2,17 (m, 2H), 1,92 is 1.75 (m, 2H). Range HRPD for the received material shown in Fig. 1. Method HPLC: column XTerra MS C18; 5 μm, a 4.6×250 mm; mobile phase: A = water fraction: 4 mm Tris, 2 mm EDTA, pH 7,4; B = organic fraction: CH3CN; gradient program: B %: 0 min 5%, 15 min 55%, 30 min 55%, 32 min 5%, 40 min 5%. Flow rate = 1 ml/min; volume of injection = 5 ál; λ: 222, 254, 282, 450 nm; retention time = 6,4 minutes

Method 3B:

Ethyl acetate (250 ml), previously cooled to 0°C, saturated with gaseous HCl for 25 minutes Add Boc-D-iGlu-D-Trp-OH (15.0 g, 34.6 mmol) with formation of a suspension. The solution is stirred for 90 minutes at a temperature of an ice bath. The solvent is evaporated in vacuum with the formation of a solid white color. The solid is dissolved in 35 ml of distilled water with the formation of a thick solution of a light brown colour. The aqueous fraction was washed with 30 ml of dichloromethane, and then transferred into a chemical beaker with a volume of 100 ml using a pH electrode to control the pH adjusted the 1,28 up 2,96 using a 3.2 ml of 10 N. NaOH solution. The solution is stirred for 1 hour at room temperature, a precipitate slowly white. The solid is separated by vacuum filtration and thoroughly washed with water. The crude solid is suspended in 20 ml of distilled water, stirring for 2 hours at room temperature. The mixture is filtered, the solid is collected and dried to constant weight in an oven under vacuum overnight (40°C). So, get 8.6 g (yield of 74.5%) specified in the connection header. Purity according to HPLC (% peak area): 98,8%. Retention time: 4,21 min; HPLC: column Waters Symmetry C 18, of 3.9×150 mm, 5 μm; mobile phase: 0.035% of HClO4pH 2/CH3CN, gradient (T [min-% CH3CN) 0-10V, 10-90, 12-90; flow rate: 1 ml/min; λ: 230, 260, 280 nm; data1H NMR answer structure.

Example 4:

The synthesis monoammonium salt D-isoglutamine-D-tryptophan (1:1) and select it using the purification column chromatography.

Method 4A:

Gaseous HCl is passed under stirring through chilled on ice (0-5°C) solution of Boc-D-Glu-(-D-Trp-OH (2.5 g, 5.8 mmol) in ethyl acetate (60 ml) for 2.5 hours. The reaction mixture is then evaporated until dry. Purification of the residue column chromatography on silica gel using a gradient solvent mixture of isopropanol and ammonium hydroxide solution (28-30% NH4H) (ratio 8:2 and 7:3, about./about.) as eluent gives named the title product (1.8 g, 84.7 per cent) in the form of a solid white color after evaporation of the solvent. TPL: 124 to 128°C;1H NMR (DMSO-d6) δ ppm: 10,98 (s, 1H), 8,25 (d, J=5,9 Hz, 1H), 7,53 (7,8 Hz, 1H), 7,30 (d, J=8.0 Hz, 1H), 7,17 (s, 1H), 7,00 (t, J=7.7 Hz, 1H), 6,92 (t, J=7.2 Hz, 1H), 4,28 (m, 1H), 3,22-of 3.31 (m, 2H), 2,90-2,96 (m, 1H), 2.23 to was 2.25 (m, 2H), 1,97-to 1.98 (m, 1H), 1,84 is 1.86 (m, 1H);13C NMR (DMSO-d6) δ ppm: 175,5 (C), 171,6 (C), 171,4 (C), 136,0 (C), uniforms, 127.6 (C), to 123.5 (CH), of 120.5 (CH), 118,3 (CH), to 117.9 (CH), 111,5 (C), 111,3 (CH), at 55.3 (CH), of 53.7 (CH), 32,5 (CH2), while 27.8 (CH2), 27,4 (CH2);14N NMR (D2O) δ ppm: 20,4 (C); IR AF (KBr) ν: 3406, 3055, 1581, 1456, 1399, 1341, 1096, 1009, 744, 535, 426 cm-1; MS (ratio of mass and charge) 334 [dibasic acid+1]+; analytical calculated for C16H22N4O5.H2O: C, 52,17; H, to 6.57; N, 15,21; found: C, 51,95; H, at 6.84; N, 14,85. The substance is monoammonium Sol D-isoglutamine-D-tryptophan (1:1). The resulting material is amorphous, which is confirmed by XRPD data.

Method 4B:

Gaseous HCl condense in cold ethyl acetate (133,4 g) at a temperature of -2oC (the temperature of an external ice bath) for 16 minutes the Increase in the mass of the solution is 21, Boc-D-iGlu-D-Trp-OH (3.8 g, 8,73 mmol) is dissolved in 50 ml of the above solution. The temperature of the mixture support 0-5°C for 55 minutes, the Reaction mixture was checked by TLC, after which evaporated when eigendom pressure (temperature rotary evaporators: 51-52°C) until dry. Purification with flash chromatography on silica gel using a gradient solvent mixture of isopropanol and ammonium hydroxide solution (28-30%) (ratio 8:2 and 7:3, vol/about.) as eluent gives the product (2.0 g, 62%) as a solid substance almost white. Data1H NMR similar to the data given in Example 4, Method 4A. The resulting material is amorphous, which is confirmed by XRPD data.

Example 5

The synthesis monoammonium salt D-isoglutamine-D-tryptophan (1:1).

Method 5A:

The synthesis monoammonium salt D-isoglutamine-D-tryptophan (1:1) by removing inorganic salts using resin AMBERLYST®15, followed by purification column chromatography.

Gaseous HCl is passed under stirring over ice (0-5°C) suspension of the obtained Boc-D-iGlu-D-Trp-OH (10,82 g, 24,96 mmol) in ethyl acetate (200 ml) for 2 hours. The reaction mixture is then evaporated until dry. The residue is dissolved in H2O (30 ml) and neutralized to pH 6-7 using 6 n NaOH solution. The resulting solution was loaded on a chromatographic column filled with resin AMBERLYST®15, followed by elution with water to pH 5-5,5, then 100% isopropanol (pH 7) and in the end a mixture of 25% NH4OH/IPA (pH 10).

The fractions that contain the product are combined and evaporated until dry under reduced pressure. Additional purification of the residue colonos the second chromatography on silica gel using a gradient mixture of solvent isopropanol and concentrated ammonium hydroxide solution (ratio of 17:3, 4:1 and 7:5, vol/about.) as eluent gives named the title product (of 6.68 g, 72,7%) as a foamy solid light yellow color. Data1H NMR and MS/MS to confirm the structure (see Methods 4A). The water content according to tests by the Karl Fischer method is 3.7%.

Method 5B:

The synthesis monoammonium salt D-isoglutamine-D-tryptophan (1:1) by removing inorganic salts using resin Amberlyst15, followed by purification by recrystallization.

When mixing through chilled on ice (0-5°C) suspension of Boc-D-iGlu-D-Trp-OH (10,75 g, 24,80 mmol) in ethyl acetate (200 ml) miss gaseous HCl. The reaction mixture is kept on ice bath (0-5°C) for 2 hours. Analysis by TLC (30% ammonia in isopropanol) shows complete conversion of the starting material. The reaction mixture is evaporated to the dry state in a vacuum, the residue is dissolved in H2O (30 ml) and neutralized to pH 6-7 with 10 N. NaOH. The obtained homogeneous solution was loaded on a chromatographic column filled with resin AMBERLYST®15, followed by elution with water (2450 ml) to pH 4-5,5, isopropanol (1000 ml) and a mixture of 25% NH4OH/isopropanol. The fractions that contain the product are combined and evaporated until dry. Get colorless foamy solid, to which is added isopropanol (150 ml) and H2O (30 ml). Received WM is enzio stirred at room temperature overnight. The solid is separated by vacuum filtration, thoroughly washed with isopropanol (2×60 ml), then EtOAc (2×60 ml) and dried in a vacuum oven at 42°C, soaking at this temperature over night. Get the solid is nearly white (6.60 g, 72.2 per cent). Data1H NMR and MS/MS reply structure (see Example 5). XRPD data obtained crystalline material is shown in Fig. 2. The water content according to Karl Fischer method was 5.9%.

Example 6

The synthesis monoammonium salt D-isoglutamine-D-tryptophan (1:1) of H-D-iGlu-D-Trp-OH.

Method 6A:

Getting monoammonium salt D-isoglutamine-D-tryptophan (1:1) using CBz-D-Glu-(γ-D-Trp-OH)-γ-OBzl as an intermediate connection.

EDC (562 mg, at 2.93 mmol) are added to a solution of Z-D-Glu-OBz (990 mg, to 2.67 mmol) and N-hydroxysuccinimide (337 mg, at 2.93 mmol) in DMF (50 ml) in an ice bath, and the resulting clear solution is stirred over night at room temperature. H-D-Trp-OH (640 mg, of 3.13 mmol) and Et3N (1 ml) is added at room temperature. After 20 minutes, the material is mixed with water and extracted with ethyl acetate. The combined EtOAc fraction was washed with 10% citric acid solution and then with saturated saline solution, dried over sodium sulfate, filtered, evaporated until dry, dried in vacuum, obtaining 1,49 g of CBz-D-iGlu-(γ-OBzl)-D-Trp-OH. The resulting material hiderou is using 33%, wt./wt. 10% Pd/C at atmospheric pressure. After 4 hours the catalyst is filtered off through CELITE® and the filtrate is evaporated to obtain an oil. The crude product was purified flash chromatography on a column using a mixture of isopropanol/NH4OH (80:20 to 70:30./about.) as eluent to obtain specified in the header of the compound (813 mg). Data MS/MS and1H NMR are similar to the compound obtained using the method shown in Example 4 above.

Method 6B:

Getting monoammonium salt D-isoglutamine-D-tryptophan (1:1) of H-D-iGlu-D-Trp-OH (see Example 3).

H-D-iGlu-D-Trp-OH (1 g from Example 3) is mixed with ammonium hydroxide (0,55 M, 6 ml). The mixture is stirred and measure the pH value, which is equal to approximately 4.5. The ammonium hydroxide solution (0,55 M) is added dropwise until the pH 7,0-7,5. Volatile materials are removed under vacuum and the residual oil is mixed with isopropanol. A white precipitate is formed. After 2 hours, the solid (ammonium salt) is separated by vacuum filtration. The solid is dried to constant weight (1 g) under a deep vacuum for 12 hours to obtain the ammonium salt of D-isoglutamine-D-tryptophan (1:1). The water content according to Karl Fischer method is 4.6%.

Example 7

Purification of H-D-iGlu-D-Trp-OH column chromatography on silica gel using a mixture of isopropanol and water.

A. the Floor is a group of Cbz-D-Glu-(γ-D-Trp-OBzl)-α-OBzl.

To a cooled on ice to a solution of 2.67 g of Cbz-D-Glu-OBzl in 50 ml of DMF type of 0.91 g of N-hydroxysuccinimide (1.1 EQ.) and 1.52 g EDC (1.1 EQ.) and the resulting solution is stirred in an ice bath for 1 hour and then at room temperature overnight. To the obtained reaction mixture was added 2.50 g of H-D-Trp-OBzl.HCl (of 1.05 equiv.) and 3 ml of Et3N at room temperature. The reaction is completed in 1 hour, which is confirmed by the HPLC data.

The reaction mixture was quenched with demineralized water in an ice bath, and then extracted several times with ethyl acetate. United an ethyl acetate extracts are washed with 10% citric acid solution and then with saturated saline solution, dried over sodium sulfate, filtered and evaporated until dry. The residue is applied to silica gel in methanol and the mixture is evaporated in vacuum. The residue is put on the upper part of the column with wet silica gel and the target product, Cbz-D-Glu-(γ-D-Trp-OBzl)-α-OBzl, elute with a gradient solvent mixture (ethyl acetate/hexane, 80:20 to 100:0). The target fractions are combined and evaporated under vacuum to obtain with 4.64 g (yield 99.6 percent) specified in the connection header. Purity according to HPLC (% peak area): 93%; HPLC conditions: column: Symmetry C18, a 3.9×150 mm, 5 μm; mobile phase: 0.035% of HClO4(pH 2,5) CH3CN = gradient (min-CH3CN %: 0-10, 10-100, 12-100, 14-50); flow rate: 1 ml/min; λ: 280 nm; in EMA retention: 9,7 minutes

B. Purification of D-isoglutamine-D-tryptophan column chromatography using a mixture of isopropanol and water.

To a suspension of 4.0 g of Cbz-D-Glu-(γ-D-Trp-OBzl)-α-Obzl obtained as described in Example 7A above, in 150 ml of a mixture of MeOH/H2O (95:5, vol/about.) add 1.5 g of Pd/C (37,5% wt./wt.). The mixture hydronaut when the hydrogen pressure of 30 lb/inch2. The reaction is terminated after 75 min, which is confirmed by the data of HPLC. The catalyst is filtered off on a layer of CELITE® and the filtrate evaporated under reduced pressure and a temperature of 45°C. Then the residue purified flash chromatography on silica gel using a mixture of isopropanol/H2O (the ratio of 80:20./vol.). The most pure fractions (according to HPLC) combined and evaporated in vacuum. Thus, get the named compound (1.1 g, 52%) as light yellow colour powder. Purity according to HPLC (% peak area): 99%. Data MS/MS and1H NMR meet the target structure.

Less pure fractions (according to HPLC) are combined, evaporated in vacuum to obtain approximately 1.0 g (yield 48%) specified in the connection header. Purity according to HPLC (% peak area): 96,7%. Data MS/MS and1H NMR correspond to the target structure.

The HPLC conditions are the same as described in section 7A above; retention time H-D-iGlu-D-Trp-OH is 4.0 minutes

Example 8

A. Obtaining H-D-Pyr-D-Trp-OH (5-oxo-D-prolyl-D-tryptophan)- D-Glu-(γ-OEt)-OH.

Specified in the header of the connection is a possible synthetic admixture of H-D-iGlu-D-Trp-OH. It is synthesized independently and used as a reference sample for analysis by HPLC of the products described in Examples 3-7 above.

To a cooled on ice to a solution of Z-D-Glu-(γ-OEt)-OH (1 g, of 3.23 mmol) in DMF (60 ml) is added N-hydroxysuccinimide (409 mg, of 3.56 mmol), EDCI (682 mg, of 3.56 mmol) and the resulting solution is stirred in an ice bath for 1 hour, then stirred at room temperature overnight. To the obtained reaction mixture was added H-D-Trp-OH (792 mg, 3.88 mmol) and Et3N (1 ml) at room temperature. Water is added over 1.5 hours at the temperature of the ice bath. The mixture is extracted several times with ethyl acetate. United an ethyl acetate extracts are washed with 10% citric acid solution, and then brine, dried over sodium sulfate, filtered, evaporated until dry and dried under vacuum to obtain 410 mg of fraction crude product A. the Aqueous fraction is evaporated in vacuum at a bath temperature of 55°C. the Residue is dissolved in CH2Cl2and the organic fraction washed with 10% citric acid solution (1×20 ml) and saturated saline solution, dried over sodium sulfate, filtered, evaporated until almost dry condition at a temperature of 50°C to obtain fractions of the crude product B. Both fra is the raw product of a and B are combined and hydronaut over 10% Pd/C (40% wt./wt. Pd/C) at atmospheric pressure using a balloon with hydrogen at room temperature for a period of 2.75 hours. The reaction mixture was filtered through CELITE®. The filtrate containing the product H-D-iGlu(γ-OEt)-D-Trp-OH, analyzed by HPLC (the same way as described in 7A above). Retention time for the peak of the product is of 4.54 min Filtrate evaporated until dry under reduced pressure at a bath temperature of about 45°C. Analysis by HPLC shows a partial transformation of the product with Rtof 4.54 min in another product with the peak of the Rtof 4.45 min the Crude product was purified flash chromatography on a column using isopropanol and concentrated solution of NH4OH (elution with a gradient from 90:10 to 80:20./vol.). Analysis by HPLC of the resulting material (670 mg) shows a peak with a value of Rt4,45 minutes Clarifying patterns1H NMR spectroscopy showed that the product after processing is a cyclic compound shown below:

1H NMR (CD3OD) δ ppm: 7,60 (d, J=7.8 Hz, 1H), 7,31 (d, J=8.0 Hz, 1H), 7,10 (s, 1H), 7,07 (t, J=7.8 Hz, 1H), 7,00 (t, J=7,4 Hz, 1H), 4,68 with 4.65 (m, 1H), 4,05-was 4.02 (m, 1H), 3,44 (DD, J=14.6 Hz, J=4.6 Hz, 1H), 3,23-3,18 (m, 1H), 2,34-of 2.24 (m, 1H), 2,13-2,05 (m, 1H), 2.00 in with 1.92 (m, 1H) and 1.76 by 1.68 (m, 1H). MS (ratio of mass and charge): 316 [M+1]+, 188 (100%).

13C NMR (CD3OD) δ ppm: 181,6, 177,5, 174,2, 138,0, 129,3, 124,5 122,4, 119,8, 119,5, 112,3, 111,7, 58,3, 56,3, 30,2, 28,8 26.5.

Method HPLC: column Symmetry C18, 5 μm, of 3.9×150 mm, WAT 046980; mobile phase - 0.035% of HClO4/CH3CN, the gradient method: min-CH3CN%: 0-10%, 10-100%, 12-100%, 14-50%; flow rate: 1.0 ml/min; detector λ: 254 nm.

B. Independent synthesis of 5-oxo-D-prolyl-D-tryptophan of (R) - (+)-2-pyrrolidin-2-5-carboxylic acid (H-D-Pyr-D-Trp-OH).

(R)(+)-2-Pyrrolidin-2,5-carboxylic acid (2,09 g to 0.016 mol) are added to 250 ml of dichloromethane. Add diisopropylethylamine (3.12 ml, 0.018 mol), and the solution becomes transparent. Hydroxybenzotriazole hydrate (HOBt.H2O, 2,48 g to 0.016 mol) is added at 0°C. Add EDCI (with 4.64 g of 0.025 mol). After 30 minutes, add H-D-Trp-OBzl.HCl (4,76 g to 0.016 mol) followed by the addition of diisopropylethylamine (3.12 ml, 0.018 mol). The solution remains clear at the point of time of 5 min, and stirred for 16 hours. The solvent is evaporated until dry, and the material is distributed between ethyl acetate and 10% chloroethanol acid. An ethyl acetate fraction was washed with 10% solution of sodium bicarbonate, then saturated saline solution. The organic fraction is dried over sodium sulfate and evaporated to obtain foamy solid. The obtained solid substance is benzyl-5-oxo-D-prolyl-D-tryptophanate. Without treatment a sample of the crude benzyl-5-oxo-D-prolyl-D-tryptophanate (1.55 g) was dissolved in methanol (3 ml) and hydronaut over 10% Pd/C under hydrogen pressure of 45 lb/in 2within 2 hours. The catalyst was filtered through celite and the filtrate is evaporated to obtain foam red. The resulting material was purified column chromatography (gradient elution: 10% methanol in dichloromethane and then 50% isopropanol in dichloromethane) to give a solid (419 mg). The purity of the material can be checked by TLC (30% aqueous solution of ammonia/isopropanol). The resulting material is a 5-oxo-D-prolyl-D-tryptophan with the same NMR data as for the material of the section A.

Example 9

The synthesis monoammonium salts of L-isoglutamine-L-tryptophan (1:1).

A: Synthesis of Boc-L-Glu-(γ-L-Trp-O-tert-Bu)-α-O-tert-Bu.

A solution of Boc-L-Glu-O-tert-Bu (1.50 g, 4.9 mmol) in CH2Cl2(50 ml) is cooled to -3°C and stirred for 15 minutes Then successively added 1-hydroxybenzotriazole (HOBt, and 1.00 g, 7.4 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC, 1.42 g, 7.4 mmol) and diisopropylethylamine (DIPEA, 1,30 ml, 7.4 mmol). Then a solution of H-L-Trp-O-tert-Bu.HCl (2.20 g, 7.4 mmol) and DIPEA (1,30 ml, 7.4 mmol) in CH2Cl2(20 ml) is added dropwise. The resulting mixture was stirred at ice temperature (-3°C to 0°C) for 1 hour and then allow to warm to room temperature and stirred overnight under nitrogen atmosphere.

The reaction mixture is evaporated to shopsonline. The residue is partitioned between EtOAc (40 ml) and a saturated solution of NaHCO3(100 ml). The organic fraction was separated, washed with 10% citric acid solution and then with saturated saline solution (30 ml). The organic fraction is dried over sodium sulfate, filtered and evaporated to a thick oil. Purification of the residue column chromatography on silica gel using a gradient solvent mixture hexane/EtOAc (85:15 ratio, 80:20 and 60:40, vol/about.) as eluent gives named the title product (2.55 g, 95%) as a solid white color.1H NMR (DMSO-d6) δ ppm: 10,84 (s, 1H), 8,18 (d, J=7.5 Hz, 1H), 7,52 (d, J=7.7 Hz, 1H), 7,35 (d, J=8.0 Hz, 1H), 7,12-to 7.15 (m, 2H), 7,06 (t, J=7,6 Hz, 1H), 6,98 (t, J=6,8 Hz, 1H), to 4.41 (q, J=6,7 Hz, 1H), of 3.73-of 3.80 (m, 1H), 2,94-of 3.12 (m, 2H), 2,13-of 2.21 (m, 2H), 1.60-to of 1.85 (m, 2H), 1,28-to 1.38 (m, 27H);13C NMR (DMSO-d6) δ ppm: which is 171,5 (C), 171,4 (C), RUB 171.1 (C), 155,5 (C), 136,1 (C), 127,2 (C), to 123.5 (CH), 120,9 (CH), 118,3 (CH), 118,1 (CH), 111,3 (CH), 109,7, 80,2, 78,0, 53,9 (CH), 53,6 (CH), 31,5 (CH2), 28,2 (CH3)of 27.9 (CH3), 27,6 (CH3), 27,5 (CH3), 27,2 (CH2), 26,6 (CH2); MS (ratio of mass and charge) 546 [M+1]+; analytical calculated for C29H43N3O7.0,5H2O: C, 62,80; H; 8,00; N, 7,58; found: C, 62,69; H, 8,56; N, EUR 7.57.

B: Synthesis monoammonium salts of L-isoglutamine-L-tryptophan (1:1).

Gaseous HCl is passed under stirring through chilled on ice (0-5°C) solution of Boc-L-Glu-(-L-Trp-O-tert-Bu)-γ-O-tert-Bu, obtained the, as described above, (2.38 g, 4.4 mmol) in CH2Cl2(40 ml) for 5 hours. The reaction mixture becomes turbid during the transmission of gaseous HCl. The reaction temperature is maintained at below 30°C by cooling on ice. The reaction mixture is then evaporated to obtain solid white (mass of raw material = 2,80 g).

Purification of the crude sample (482 mg) using reversed-phase high performance flash chromatography (HPFC™, Biotage) using column C18HS M+40 and a gradient solvent mixture of 15 mm solution of NH4OAc/CH3CN as eluent gives the titled compound after evaporation of the solvent and freeze drying material (150 mg).1H NMR (DMSO-d6) δ ppm: EUR 7.57 (d, J=7.8 Hz, 1H), 7,39 (d, J=8,1 Hz, 1H), 7,11-7,14 (m, 2H), 7,05 (t, J=7.2 Hz, 1H), of 4.44-4,48 (m, 1H), 3,42 (t, J=5.7 Hz, 1H), 3,25 (DD, J=14,7, a 4.7 Hz, 1H), 2,97-3,03 (m, 1H), 2,14-2,19 (m, 2H,), 1,72-of 1.85 (m, 2H); IR AF (KBr) ν: 3057, 1581, 1400, 745 cm-1; MS (ratio of mass and charge) 334 [dibasic acid+1]+; analytical calculated for C16H22N4O5.H2O: C, 52,17; H, to 6.57; N, 15,21; found: C, 51,92; H, to 6.80; N, 14,94. The substance is monoammonium salt L-isoglutamine-L-tryptophan (1:1).

Example 10

The synthesis monoammonium salt of H-D-isoglutamine-L-tryptophan (1:1).

A. Synthesis of Boc-D-Glu-(γ-L-Trp-O-tert-Bu)-α-O-tert-Bu.

Use the methods described in Example 1A. When stirring is cooled on ice to a solution of Boc-D-Glu-O-tert-Bu (4,00 g, 13,2 mmol) in CH2Cl2(75 ml) was successively added EDC (3.80 g, and 19.8 mmol), HOBt (2,68 g of 19.8 mmol) and DIPEA (3,50 ml of 19.8 mmol). The resulting mixture was stirred at ice temperature for 20 minutes Then a solution of H-L-Trp-O-tert-Bu.HCl (5,88 g of 19.8 mmol) and DIPEA (3,50 ml of 19.8 mmol) in CH2Cl2(50 ml) is added dropwise within 10 minutes the resulting mixture was stirred at ice temperature for 1 hour, then allow to warm to room temperature and stirred overnight.

The reaction mixture is evaporated until dry. Thick residual oil is transferred into ethyl acetate (50 ml) and the organic fraction washed subsequently with a saturated solution of NaHCO3(100 ml), 10% citric acid solution (100 ml), saturated brine (100 ml) and water (100 ml). The organic fraction is dried over sodium sulfate, filtered and evaporated in vacuum. Purification of the residue column chromatography on silica gel using a gradient solvent mixture hexane/EtOAc (8:2 ratio of 7:3 and 6:4.about.) as eluent gives named the title product (5,88 g, 82%) as a solid substance almost white.1H NMR (DMSO-d6) δ ppm: 10,85 (s, 1H), 8,18 (d, J=7,4 Hz, 1H), 7,50 (d, J=7.7 Hz, 1H), 7,33 (d, J=8.0 Hz, 1H), 7,14-7,05 (m, 3H), of 6.99 (t, J=7,1 Hz, 1H), and 4.40 (q, J=7.5 Hz, 1H), 3,81 of 3.75 (m, 1H), 3,12-of 3.07 (m, 1H), 3,01-2,95 (m, 1H), 2,18-of 2.15 (m, 2H), 1,86 of-1.83 (m, 1H), 1,73-of 1.65 (m, 1H), 1,39-of 1.29 (m, 27H); MS (is the rate of mass and charge) 568 [M+Na] +; 546 [M+1]+; analytical calculated for C29H43N3O7.0,75H2O: C, 62,29; H, 8,02; N, 7,51. Found: C, 62,43; H, Of 7.95; N, 7,08.

B. Synthesis monoammonium salt of H-D-Glu-(-L-Trp-OH (1:1).

Use the methods described in Example 1B. Gaseous HCl is passed under stirring through chilled on ice (approximately -5°C) solution of Boc-D-Glu-(γ-L-Trp-O-tert-Bu)-α-O-tert-Bu, obtained as described above (1,59 g, only 2.91 mmol) in ethyl acetate (100 ml) for 45 minutes the Solution becomes colorless dull yellow. The mixture is stirred at ice temperature for 1 hour, then allow to warm to room temperature and stirred for another 2 hours. According to HPLC, the reaction is finished (column: Waters C18, a 3.9×150 mm, WAT046980, mobile phase: gradient solvent mixture 0.035% of HClO4[pH 2-2,5]/acetonitrile, flow rate: 1 ml/min, 8: 210-270 nm).

The reaction mixture is evaporated under reduced pressure to obtain a solid substance. The solid is dissolved in acetone and volatile components removed under reduced pressure. The last procedure is repeated twice more. Purification of the residue column chromatography using a gradient solvent mixture of isopropanol and ammonium hydroxide solution (28-30% NH4OH) (ratio 85:15 and 70:30, vol/about.) as eluent gives named the title product (0,42 g, 39%) as a foam almost be the CSOs color. TPL: 120-130°C;1H NMR (D2O) δ ppm: to 7.67 (d, J=7.9 Hz, 1H), 7,46 (d, J=8,2 Hz, 1H), 7.18 in-7,22 (m, 2H), 7,13 (t, J=7,1 Hz, 1H), 4,53 (kV, J=3.8 Hz, 1H), 3.45 points (t, J=5.8 Hz, 1H), 3,33 (DD, J=14.7 and of 4.75 Hz, 1H), of 3.07 (DD, J=14,7 and 8.8 Hz, 1H), 2,19-2,31 (m, 2H), 1,78-to 1.98 (m, 2H);13C NMR (D2O) δ ppm: 181,4 (C), 176,6 (C), 176,5 (C), 138,8 (C), 129, 9mm (C), 126,9 (CH), 124,5 (CH), 121,9 (CH), 121,4 (CH), of 114.5 (CH), of 113.2 (C), to 58.6 (CH), of 56.7 (CH), 34,2 (CH2), 30,3 (CH2), to 28.9 (CH2); MS (ratio of mass and charge) 334 [dibasic acid+1]+.

Example 11

The synthesis monoammonium salts of L-isoglutamine-D-tryptophan (1:1).

A. Synthesis of Boc-L-Glu-((-D-Trp-OMe)-α-O-tert-Bu.

Use the methods described in Example 1A. With stirring to a cooled on ice to a solution of Boc-L-Glu-O-tert-Bu (3,45 g of 11.4 mmol) in CH2Cl2(120 ml) was successively added EDC (of 3.31 g, 17.3 mmol), HOBt (2,36 g, 17.5 mmol) and DIPEA (3.0 ml, of 17.1 mmol). The resulting mixture was stirred at ice temperature for 25 minutes Then add a solution of H-D-Trp-OMe.HCl (2.20 g, 7.40 mmol) and DIPEA (3.0 ml, of 17.1 mmol) in CH2Cl2(40 ml). The resulting mixture was stirred at ice temperature for 1 hour, then allowed to warm to room temperature and stirred overnight.

After conventional treatment, as described in Example 1A, purification of the residue column chromatography using a gradient solvent mixture hexane/EtOAc (ratio 8:2 and 6:4, by vol./about.) as eluent gives named the title product is t (4,25 g, 74%) as a white foam.1H NMR (CDCl3) δ ppm: 8,64 (s, 1H), 7,53 (d, J=7.7 Hz, 1H), 7,33 (d, J=8.0 Hz, 1H), 7,15 (t, J=7.2 Hz, 1H), was 7.08 (t, J=7.5 Hz, 1H), 6,98 (s, 1H), is 6.61 (d, J=7.2 Hz, 1H), and 5.30 (d, J=7.8 Hz, 1H), 4.92 in (kV, J=6,8 Hz, 1H), 4,16-4,17 (m, 1H), to 3.67 (s, 3H), 3,28-to 3.34 (m, 2H), 2,16-of 2.27 (m, 2H), 2.05 is with 2.14 (m, 1H), 1,79-1,89 (m, 1H), 1,42 was 1.43 (m, 18H);13C NMR (CDCl3) δ ppm: 172,6 (C), 172,1 (C), 171,6 (C), is 171.3 (C), 155,9 (C), 136,3 (C), of 127.7 (C)of 123.2 (CH), 122,2 (CH), 119,6 (CH), 118,6 (CH), 111,5 (CH), 109,9 (C)82,3 (C), 79,9 (C)of 53.7 (CH), 53,3 (CH), a 52.4 (CH), 32,6 (CH2), to 28.9 (CH2), 28,4 (CH3), 28,1 (CH3), with 27.7 (CH2); MS (ratio of mass and charge) 504 [M+1]+; analytical calculated for C26H37N3O7.0,25H2O: C, 61,46; H, 7,44; N, of 8.27; found: C, 61,36; H, 7,50; N, 7,84.

B. Synthesis of Boc-L-iGlu-D-Trp-OH.

With stirring to a solution of Boc-L-Glu-((-D-Trp-OMe)-α-O-tert-Bu (3.94 g, 7.82 mmol) in methanol (50 ml) add a solution of NaOH (654 mg, 16.4 mmol) in H2O (20 ml). The resulting solution was stirred at room temperature overnight. 1 N. NaOH Solution (150 ml) are added to the reaction mixture and the aqueous material is washed with ethyl acetate (3×100 ml). The aqueous fraction is acidified with 3 N. a solution of HCl to approximately pH 2, then extracted with ethyl acetate (3×100 ml). The organic fractions combined, dried over sodium sulfate and evaporated under reduced pressure. Purification of the residue column chromatography on silica gel using a gradient mixture of solvent CH2Cl2and MeOH (sootnoshenie is 85:15, 70:30, vol/about.) as eluent gives named in the title of the product (0.55 g, 97%) as a pink foam.1H NMR (MeOD-d4) δ ppm: 7,58 (d, J=7.7 Hz, 1H), 7,31 (d, J=8.0 Hz, 1H), 7,05-to 7.09 (m, 2H), 6,99 (t, J=7,3 Hz, 1H), br4.61-of 4.67 (m, 1H), 4,13 (W, 1H), 3,30-to 3.38 (m, 2H), 3,12-3,18 (m, 1H), 2.21 are of 2.27 (m, 2H), 1,98-to 2.06 (m, 1H), 1,81-of 1.88 (m, 1H), of 1.42 (s, 9H);13C NMR (MeOD-d4) δ ppm: 158,2 (C), 138,1 (C), 129,1 (C)124,5 (CH), 122,4 (CH), 119,9 (CH), 119,5 (CH), 112,3 (CH), 111,5 (C), 80,6 (C)33,5 (CH2), to 28.9 (CH3), or 28.7 (CH2); MS (ratio of mass and charge) 490 [M + 1]+.

C. Synthesis monoammonium salt H-L-iGlu-D-Trp-OH (1:1).

Gaseous HCl is passed under stirring through chilled on ice (approximately 0°C) solution of Boc-L-iGlu-D-Trp-OH as described above (500 mg, 1.0 mmol) in a mixture of solvents CH2Cl2(20 ml) and EtOAc (10 ml) for 30 min. the Reaction mixture was stirred at ice temperature for 1 hour. According to HPLC, the reaction is finished (column: Waters C18, a 3.9×150 mm, WAT046980, mobile phase: gradient solvent mixture 0.035% of HClO4[pH 2.0 to 2.5)/acetonitrile, flow rate: 1 ml/min, 8: 210-270 nm).

The reaction mixture is evaporated to the dry state with obtaining foam deep purple color. Purification of the residue column chromatography using a gradient solvent mixture isopropanol/ammonium hydroxide solution (28-30% NH4OH) (ratio 85:15 and 70:30, vol/about.) as eluent gives named the title product (333 mg, 88%) in VI is e thick orange oil. 1H NMR (D2O) δ ppm: to 7.64 (d, J=7.9 Hz, 1H), 7,44 (d, J=8.0 Hz, 1H), 7,16-7,19 (m, 2H), 7,10 (t, J=7.5 Hz, 1H), to 4.52 (q, J=3,7 Hz, 1H), 3,44 (t, J=6.3 Hz, 1H), 3,29-to 3.34 (m, 1H), 3,03-to 3.09 (m, 1H), 2,17-of 2.30 (m, 2H), 1,76 is 1.96 (m, 2H);13C NMR (D2O) δ ppm: 181,2 (C), 176,6 (C), 176,5 (C), 138,8 (C), 129, 9mm (C), 126,9 (CH), 124,5 (CH), 121,9 (CH), 121,4 (CH), of 114.5 (CH), 113,1 (C)58,5 (CH), of 56.7 (CH), 51,6 (CH), 34,2 (CH2), 30,2 (CH2), to 28.9 (CH2); MS (ratio of mass and charge) 356 [dibasic acid+Na]+, 334 [dibasic acid+1]+, analytically calculated for C16H22N4O5.2,35H2O: C, 48,94; H, 6,85; N, 14,27; found: C, 48,94; H, only 6.64; N, of 14.28.

Example 12

Analysis by HPLC D-isoglutamine-D-tryptophan, L-isoglutamine-L-tryptophan, L-isoglutamine-D-tryptophan and D-isoglutamine-L-tryptophan.

Dibasic acid of the four diastereomers were analyzed on a chiral HPLC column. D,D - and L,D-diastereoisomers taken from the examples above, while D,L-isomer obtained from Bachem and L,L-isomer obtained from Sigma. Analysis using chiral column HPLC (table. 1) showed that the obtained D-isoglutamine-D-tryptophan is free of other diastereomers, namely (D,L), (L,L) and (L,D) isomers.

Table 1
Analysis of H-iGlu-Trp-OH by HPLC
H-iGlu-Trp-OHRetention time (HPLC, Method A, min)Time hold the cation (HPLC, Method B, min)
(D,D) diastereoisomer19,323,92
(D,L) diastereoisomer9,463,92
(L,D) diastereoisomer13,99a 3.87
(L,L) diastereoisomer6,703,91

Method a:
Column:CHIROBIOTIC® TAG, 5 μm, a 4.6×250 mm
Mobile phase:20 mm solution of ammonium acetate (pH of 4.1)/MeOH (80:20)
Flow rate:0.8 ml/min
The detector, λ:222, 254, 282, 450 nm
The temperature of the column:45°C
Method:
Column:Symmetry C18, part No. WAT 046980
Mobile phase:HClO4(pH 2)/CH3CN (85/15
Flow rate:1.0 ml/min
The detector, λ:210-280 nm

In the method And the samples analyzed on a chiral column. The values of the retention time for all four diastereomers is obviously different. In method B, the samples will be analyzed by conventional reversed-phase column; there is practically no difference in retention time for samples. Monoamina Sol D-isoglutamine-D-tryptophan disclosed in this invention, is stable after 2 years of storage. Analysis by HPLC (method B) showed that the purity at a wavelength of 254 nm is 99,8%.

Example 13

A. obtaining a salt of H-D-Glu-(γ-D-Trp-OMe)-α-OBzl HCl {(2R)-amino-(4R)-[2-(1H-indol-3-yl)-1-methoxycarbonyl-ethylcarboxyl]-butyric acid benzyl ester hydrochloride}.

A 3-necked round bottom flask of 250 ml, equipped with a magnetic stirrer, place of Boc-D-Glu-(γ-D-Trp-OMe)-α-OBzl (20 g, 0,037 mol) and 100 ml of dichloromethane with the formation of a clear solution with stirring. The solution is cooled in a cooling bath containing a mixture of ice/NaCl, up to -10°C. Gaseous HCl is bubbled through the cold solution. During the reaction temperature ranges from -4°C to -10°C. the Reaction is terminated after approximately 1 hour. Solid white precipitates and the solution. Product in the form of a solid substance separated by filtration. The solid is washed with dichloromethane (2×40 ml), dried first in air, then dried in a vacuum oven at a temperature of 42°C overnight with the receipt of 16.4 g (94%, purity according to HPLC to 98.2%).1H NMR (DMSO-d6) δ ppm: of 10.93 (s, 1H), 8,61 (Shir, 3H), and 8.50 (d, J=7,4 Hz, 1H), 7,47 (d, J=7,4 Hz, 1H), 7,40-to 7.32 (m, 6H), 7,17 (s, 1H), 7,06 (t, J=7,4 Hz, 1H), 6,97 (t, J=7,4 Hz, 1H), 5,26-5,14 (m, 2H), 4,51-to 4.46 (m, 1H), 4,05-3,95 (m, 1H), of 3.56 (s, 3H), 3,16-3,11 (m, 1H), 3,06-a 3.01 (m, 1H), 2,40-of 2.26 (m, 2H), 2.00 in to 1.98 (m, 2H). Method HPLC: column: XTerra MS C18 5 μm to 4.6×250 mm; mobile phase: A = water phase: 4 mm Tris, 2 mm EDTA, pH 7,4, B = organic phase: CH3CN. The gradient program: B %: 0 min 5%, 15 min 55%, 30 min 55%, 32 min 5%, 40 min 5%. Flow rate: 1 ml/min; volume of injection = 5 μl; wavelength: 222, 254, 282, 450 nm. Rtthe source material = 25,1 min; Rtproduct = 17,2 minutes

B. Obtain (2R)-amino-(4R)-[2-(1H-indol-3-yl)-1-methoxycarbonyl-ethylcarboxyl]-butyric acid methyl ester hydrochloride salt of H-D-Glu-(γ-D-Trp-OMe)-α-OMe HCl.

A 3-necked round bottom flask of 250 ml, equipped with a magnetic stirrer, place of Boc-D-Glu-(γ-D-Trp-OMe)-α-OMe (2.8 g, the 6.06 mmol) and methanol (30 ml) to give a clear solution with stirring. The solution is cooled in a cooling bath containing a mixture of ice/NaCl, and -12°C. Gaseous HCl is bubbled through the cold solution. During the reaction temperature is in the range from -12°C on the +9°C. The reaction is terminated after approximately 30 minutes Approximately half of the reaction mixture is evaporated until dry to obtain 1.3 g of a solid substance (purity according to HPLC 96.8 per cent).1H NMR (DMSO-d6) δ ppm: 10,90 (s, 1H), 8,48-8,46 (m, 4H), of 7.48 (d, J=7.8 Hz, 1H), 7,34 (d, J=8.0 Hz, 1H), 7,16 (s, 1H), 7,07 (t, J=7,4 Hz, 1H), 6,98 (t, J=7,4 Hz, 1H), to 4.52-4,47 (m, 1H), 4,05-to 3.99 (m, 1H), 3,69 (s, 3H), to 3.58 (s, 3H), 3,17-of 3.12 (m, 1H), 3,07-a 3.01 (m, 1H), 2,37-of 2.23 (m, 2H), 1,98 is 1.91 (m, 2H). Method HPLC: column: XTerra MS C18 5 μm to 4.6×250 mm; mobile phase: A = water phase: 4 mm Tris, 2 mm EDTA, pH 7,4; B = organic phase: CH3CN. The gradient program: B %: 0 min 5%, 15 min 55%, 30 min 55%, 32 min 5%, 40 min 5%. Flow rate: 1 ml/min Volume of injections = 5 μl; wavelength: 222, 254, 282, 450 nm; Rtthe source material = 18,7 min, Rtproduct = 13,0 minutes

C. Obtaining salt of H-D-Glu-(γ-D-Trp-OMe)-α-OBzl HCl {(2R)-amino-(4R)-[2-(1H-indol-3-yl)-1-methoxycarbonyl-ethylcarboxyl]-butyric acid benzyl ester hydrochloride}.

A solution of HCl in ethyl acetate is produced by passing gaseous HCl formed by adding dropwise 50 ml conc. HCl to end. H2SO4through chilled on ice (0-4°C) ethyl acetate (100 ml). Received a cold solution of the acid is placed in the addition funnel and added dropwise to a thin suspension of Boc-D-Glu(D-Trp-OMe)-OBzl (36,45 g, 67,80 mmol) in 150 ml of ethyl acetate. Thick suspension formed within 5 minutes Then add the HCl solution, gaseous HCl, as described above, and passed directly through the resulting suspension. The internal temperature of the support in the range of 5-10°C and cooling on ice. With stirring, the temperature of the suspension is maintained at a level of 5-15°C, soaking at this temperature for 2.5 hours, then allow to warm to room temperature. The course of the reaction is controlled by TLC (hexane/EtOAc [1:1 vol./about.] as eluent). The solid is collected by vacuum filtration, washed with ethyl acetate (2×100 ml), then dried in a vacuum oven over night. Get a solid light pink color (output 27,49 g, 86%). Analytical data similar to that shown in Example 13A above, except a certain amount of residual EtOAc detected according to the1H NMR. Residual EtOAc can be removed by drying in air for 24 to 48 hours before drying in a vacuum oven.

D. Obtaining salt of H-D-Glu-(γ-D-Trp-OMe)-α-OBzl HCl {(2R)-amino-(4R)-[2-(1H-indol-3-yl)-1-methoxycarbonyl-ethylcarboxyl]-butyric acid benzyl ester hydrochloride}

A 3-necked round-bottom flask Boc-D-Glu-OBzl (200,0 g, 0,593 mol) and EtOAc (1.6 l). Diisopropylethylamine (206,5 ml, 1,186 mol) are added to the resulting suspension white. The internal temperature is approximately 21°C, and there is a thick suspension. The suspension gradually razreshaetsja Yves within 15 minutes turns into a clear solution. Next, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (to 136.4 g of EDC, 0,711 mol) was added as a suspension. HCl.H-D-TrpOMe (166,1 g, 0,652 mol) is added in portions within 30 minutes There is heat, and the internal temperature reaches 28°C. After 2 hours, the internal temperature is reduced to 25°C. the resulting suspension is vigorously stirred over night at room temperature. The reaction mixture was diluted with ethyl acetate (200 ml), then successively washed with 1 N. HCl (600 ml) and 5% HCl solution (2×256 ml). A sample of the crude mixture evaporated and analyzed by TLC and1H NMR. The water content according to Karl Fischer method is 2.3%.

The contents of the flask cooled in an ice bath, and the internal temperature is approximately 0°C. Gaseous HCl is passed through a chilled on ice the solution for 1 hour. There is heat within 10 minutes after passing gaseous HCl, and the internal temperature is approximately 19°C. After 20 min bandwidth internal temperature is 22°C. the resulting suspension is stirred for another hour. The solid is separated by vacuum filtration and washed with ethyl acetate (2×640 ml), then dried under vacuum to constant weight (243,8 g). Analytical data similar to that shown in Example 13A above. The presence of the ethyl acetate is (approximately 10% according to the proton integration) confirmed 1H NMR. Captured EtOAc can be removed by drying the solids in the air in 24-48 hours before drying in the oven.

Example 14

Obtaining solution for major additive salts of D-isoglutamine-D-tryptophan, and its transformation in thymodepressin (D-isoglutamine-D-tryptophan).

Methodology 14A:

Source material, Sol H-D-Glu-(γ-D-Trp-OMe)-α-OBzl HCl (4.0 g, 8.4 mmol) is placed in a 3-necked round bottom flask of 250 ml, equipped with a magnetic stirrer. Add methanol (20 ml) to obtain a transparent solution. The solution is cooled in a bath containing ice and salt, NaCl, to -10°C. Add a solution of NaOH (3 N., and 8.4 ml of 25.2 mmol). HPLC is used to monitor the progress of the reaction. After 2 hours the analysis of the reaction mixture by HPLC shows that the reaction is not yet finished. Add a solution of NaOH (3 N., 1,4 ml, 4.2 mmol). At this point add in General, 29.4 mmol NaOH. After another 4 hours analysis of the reaction mixture by HPLC shows that the content of the product in the reaction mixture is more than 95.2 per cent. The reaction is stopped. The reaction mixture is acidified while cooling with a bath containing ice water until a pH of 6.5 by adding chloroethanol acid (6 N., ~1.3 ml, 7.8 mmol). The resulting solution was evaporated to a volume of 15 ml to remove most of the methanol. The solution is washed with ethyl acetate (15 ml × 2). The solution is filtered and collect the filtrate. The filtrate additionally padcal the Ute to pH 3 by adding chloroethanol acid (6 N., ~1.3 ml, 7.8 mmol). At this point, use the total number of ~15.6 mmol chloroethanol acid. The solid formed during stirring at room temperature. The mixture is stirred over night. The solid is separated by filtration. The solid is air-dried to obtain 2.4 g of crude product. Next, the solid is again placed in a round bottom flask. Add demineralized water (15 ml) and the mixture is stirred for 2 hours. The solid is separated by filtration. The solid is then dried in air, and then placed in a round bottom flask. Add demineralized water (15 ml) and the mixture is stirred for 1 hour. The solid is separated by filtration and washed with ice demineralized water (6 ml × 3). By testing with silver nitrate it is shown that the solid does not contain chlorides. The solid is air-dried, then placed in a vacuum oven at a temperature of 42°C, maintaining at this temperature for 19 h, to obtain 1.3 g (46%, purity according to HPLC 98,8%). The filtrate and aqueous solutions for rinsing combined and evaporated to allocate a second portion of the product.1H NMR (D2O-NaOD, pH 7.0) δ ppm: to 7.59 (d, J=7,6 Hz, 1H), 7,38 (d, J=7,6 Hz, 1H), 7,15 for 7.12 (m, 2H), 7,05 (t, J=7.2 Hz, 1H), 4,47-of 4.44 (m, 1H), 3,40 (t, J=6,1 Hz, 1H), 3,30-of 3.25 (m, 1H), 3,03-of 2.97 (m, 1H), 2,3-2,1 (m, 2H), 1,84 was 1.69 (m, 2H). MS (soo is wearing mass and charge) 334,3 [M+1] +. Method HPLC: column: XTerra MS C18 5 μm to 4.6×250 mm Mobile phase: A = water phase: 4 mm Tris, 2 mm EDTA, pH 7,4; B = organic phase: CH3CN. The gradient program: B %: 0 min 5%, 15 min 55%, 30 min 55%, 32 min 5%, 40 min 5%. Flow rate: 1 ml/min Volume of injections = 5 ál. Wavelength: 222, 254, 282, 450 nm. Rtproduct = 6,5 minutes

Methodology 14B:

The monohydrate of lithium hydroxide (0,374 g, 8.9 mmol) dissolved in 3.5 ml of demineralized water. The solution is placed in odnogolosy a round bottom flask of 100 ml equipped with a magnetic stirrer. 6,5 ml of Methyl tert-butyl ether is added to the solution. At room temperature add the source material, Sol H-D-Glu-(γ-D-Trp-OMe)-α-OBzl HCl (2.0 g, 4.2 mmol) with formation of a suspension. Add methanol (2 ml), most of the solids dissolved. HPLC is used to control the reaction. A number of the original material remains in the reaction mixture after stirring at room temperature over night. Lithium hydroxide monohydrate (0,190 g, 4.5 mmol) dissolved in 2 ml of demineralized water and added to the reaction mixture, followed by adding 2 ml of methanol. At this point add the total number of 13.4 mmol LiOH. After taking a 4 hour analysis of the reaction mixture by HPLC shows that the reaction was not yet complete. The monohydrate of lithium hydroxide (0,100 g, 2.4 mmol) dissolved in 1 ml of dem is maralitang water and add to the reaction mixture. At this point add the total amount to 15.8 mmol LiOH. After another 2.5 hours analysis of the reaction mixture by HPLC shows that the content of the product in the reaction mixture exceeds 97.5 per cent. The reaction is stopped. The reaction mixture was poured into a separating funnel and 2 of the faction divide. The aqueous fraction is washed with ethyl acetate (15 ml × 2). The aqueous fraction is acidified while cooling with a bath containing water with ice, to pH 6 by adding chloroethanol acid (6 N., ~650 μl, 3.9 mmol). The aqueous fraction is evaporated to 5 ml and filtered, collecting the filtrate. The filtrate is optionally acidified to pH 3 by adding chloroethanol acid (6 N., ~700 μl, 4.2 mmol). At this point, use the total number of ~8.1 mmol chloroethanol acid. The solid formed during stirring at room temperature. The solid is separated by filtration. The solid is air-dried, then placed in a round bottom flask. Add demineralized water (6 ml) and the mixture is stirred for 15 minutes, the Solid is separated by filtration and washed with ice-cold demineralized water (6 ml × 6). Informed that the solid does not contain chlorides, by testing with silver nitrate. The solid is air-dried, then placed in a vacuum oven at a temperature of 42°C, soaking at this temperature for the of 12 hours, with the receipt of 0.44 g (31%, purity according to HPLC 98.5 per cent). The filtrate and aqueous solutions for rinsing combined and evaporated to an additional allocation of the second portion of the product.1H NMR (D2O-NaOD, pH 6,0) δ ppm: to 7.59 (d, J=7.7 Hz, 1H), 7,38 (d, J=7.7 Hz, 1H), 7,15 for 7.12 (m, 2H), 7,05 (t, J=7.2 Hz, 1H), 4,47-of 4.44 (m, 1H), 3,41 (t, J=6.0 Hz, 1H), 3,29-of 3.25 (m, 1H), 3,03-of 2.97 (m, 1H), 2,3-2,1 (m, 2H,), 1,83 is 1.58 (m, 2H).

Method HPLC: column: XTerra MS C18 5 μm to 4.6×250 mm Mobile phase: A = water phase: 4 mm Tris, 2 mm EDTA, pH 7,4; B = organic phase: CH3CN. The gradient program: B %: 0 min 5%, 15 min 55%, 30 min 55%, 32 min 5%, 40 min 5%. A flow rate of 1 ml/min Volume of injections = 5 ál. Wavelength: 222, 254, 282, 450 nm. Rtproduct = 6,5 minutes

Thymodepressin obtained in this invention has a solubility in water of from about 20 to about 23 mg/ml of the Washing water in the Methods A and B serves to remove inorganic salts such as sodium chloride or lithium chloride. When receiving large amounts of volume of water for washing can be controlled by computing a number of inorganic salts present and using solubility to determine the amount of water necessary to wash the product.

As can be implemented numerous changes in preferred variants of the present invention without departing from the context of the present invention, it is envisaged that all mater is al, included in this description should be interpreted as illustrating the invention and not limiting it.

1. Crystalline D-isoglutamine-D-tryptophan, which is characterized by peaks in the powder x-ray with the following 2θ values: 18,87, 20,05, 23,74 and 29,91.

2. Crystalline D-isoglutamine-D-tryptophan, which is characterized by a powder x-ray shown in figure 1.

3. Crystalline D-isoglutamine-D-tryptophan, which is characterized by the following parameters of powder x-ray presented in values of interplanar distance d, Bragg angle 2θ and relative intensity (expressed as a percentage relative to the most intense line):

Angle °2θThe value of d, ARelative intensity [%]
to 6.6713,2393
11,097,9754,4
11,777,5151,2
to 13.296,6554
14,266,25 11,3
15,585,68533,3
the value of 16,815,26928,9
17,275,1330,4
18,354,83212,2
18,87the 4.795,8
20,05at 4,42463,6
20,94,24733,2
22,034,03217,1
22,88of 3.884100
23,743,74497,9
24,543,62541,9
25,443,49920,3
25,693,46512,1
26,31 3,38416,4
273,327,4
27,753,21224,9
as opposed to 28.18 per3,16419,3
28,793,0996,8
29,133,0636,2
29,912,98579,2
31,042,8798,6
31,492,83933,7
32,542,7494,4
33,292,6899,3
33,972,63710,5
34,992,562the 17.3
35,542,52421,8
36,14 2,4835,1
36,742,4445,9
37,352,4067,7
38,312,34825,6
39,012,30720,3

4. The method of obtaining D-isoglutamine-D-tryptophan, free from inorganic salts, comprising the following stages:
(a) obtaining a solution of an acid additive salt of H-D-iGlu-D-Trp-OH in aqueous medium essentially free of organic solvent; or the receiving solution is basically additive salt of H-D-iGlu-D-Trp-OH in aqueous medium essentially free of organic solvent;
(b) correction of the pH to values in the range from approximately 2.0 to approximately a 3.2 with a solution of a hydroxide of an alkali metal or inorganic acid to cause precipitation of H-D-iGlu-D-Trp-OH;
(c) isolation of the precipitated H-D-iGlu-D-Trp-OH; and
(d) vacuum drying the product obtained in stage (C) obtaining H-D-iGlu-D-Trp-OH.

5. The method according to claim 4, in which the acid additive salt is a H-D-iGlu-D-Trp-OH hydrochloride obtained by the process comprising the following stages:
(i) basic hydrolysis of the compounds of formula I:

where R1selected from the group consisting of C1-C4of alkyl and benzyl, and R2represents a C1-C4alkyl, provided that C4alkyl is tert-bootrom,
using a metal hydroxide in water and an inert solvent, in the presence of methanol to obtain Boc-D-iGlu-D-Trp-OH, free of other diastereomers; and
(ii) removal using hydrogen chloride protection with Boc-D-iGlu-D-Trp-OH from the stage (i) in an inert organic solvent; and evaporating the solvent to obtain hydrochloride H-D-iGlu-D-Trp-OH.

6. The method according to claim 4, wherein the acid additive salt is a H-D-iGlu-D-Trp-OH hydrochloride, and the solution of acid additive salt at the stage of (a) receiving method including:
(i) hydrogenation of the compounds of formula II

where R1represents benzyl, and R2selected from the group consisting of benzyl, and hydrogen using palladium on coal in methanol or ethanol;
(ii) purification of crude H-D-iGlu-D-Trp-OH from the stage (i) using chromatography on silica gel using isopropanol and water as an eluent; and (iii) processing of material from step (ii) chloroethanol acid in water to obtain a solution of hydrochloride salt of H-D-iGlu-D-Trp-OH in water.

7. The method according to claim 4, in which the solution is basically additive salt of H-D-iGlu-D-Trp-OH in stage (a) receive ways the Ohm, including:
(i) removal using acid-protected dipeptide Boc-D-Glu-(γ-D-Trp-OR2)-α-OR1obtaining an acid additive salt diapir H-D-Glu-(γ-D-Trp-OR2)-α-OR1where each of R1and R2independently selected from the group consisting of C1-C4of alkyl and benzyl;
(ii) basic hydrolysis of the product from step (i) with a metal hydroxide in water and an inert solvent in the presence of methanol to obtain basic additive salt of H-D-iGlu-D-Trp-OH, where R1and R2are as defined above, and where the hydroxide of a metal selected from the group consisting of sodium hydroxide, potassium hydroxide and lithium hydroxide;
(iii) extraction of the material from step (ii) in a solvent not miscible with water, and separating the aqueous layer;
(iv) the correction of the pH of the aqueous phase from stage (iii) to a pH from about 6 to about 7; and
(v) evaporating the solvent from the solution from step (iv) with the formation of a solution containing approximately one part solute to less than about 8 parts of water, where the dissolved substance is a basic additive salt of D-isoglutamine-D-tryptophan.

8. The way of getting free from inorganic salts H-D-iGlu-D-Trp-OH according to claim 4, which includes:
(a) obtaining a solution of an acid additive salt of H-D-iGlu-D-Trp-OH in aqueous medium essentially free from the solvent content of inorganic fillers;
(b) correction of the pH to values of from approximately 2.0 to approximately a 3.2 with a solution of alkali metal hydroxide, to cause the precipitation of H-D-iGlu-D-Trp-OH;
(c) isolation of the precipitated H-D-iGlu-D-Trp-OH; and
(d) vacuum drying the product obtained in stage (C), obtaining H-D-iGlu-D-Trp-OH.

9. The way of getting free from inorganic salts H-D-iGlu-D-Trp-OH according to claim 4, which includes:
(a) obtaining a solution of a basic additive salt of H-D-iGlu-D-Trp-OH in aqueous medium essentially free of organic solvent;
(b) correction of the pH to values of from approximately 2.0 to approximately a 3.2 with a solution of inorganic acid to cause precipitation of H-D-iGlu-D-Trp-OH;
(c) isolation of the precipitated H-D-iGlu-D-Trp-OH; and
(d) vacuum drying the product obtained in stage (C), obtaining H-D-iGlu-D-Trp-OH.

10. The method of claim 8 in which the acid additive salt on the stage (a) is a H-D-iGlu-D-Trp-OH hydrochloride.

11. The method according to claim 8 or 9, in which the pH at the stage (b) is from approximately 2.5 to approximately 3.0.

12. The method according to claim 5, in which R1represents benzyl, and R2represents methyl.

13. The method according to claim 5 in which the inert solvent in stage (i) is selected from the group consisting of methanol and methyl tert-butyl ether; and a hydroxide of a metal selected from the group consisting of sodium hydroxide, lithium hydroxide and hydro is sid potassium.

14. The method according to claim 9, in which in the compound of the formula Boc-D-Glu-(γ-D-Trp-OR2)-α-OR1at the stage of (a) R1selected from the group consisting of methyl and benzyl, and R2selected from the group consisting of methyl and ethyl.

15. The method according to claim 9 in which the inert solvent at the stage (b) is selected from the group consisting of methanol and methyl tert-butyl ether.

16. Crystalline ammonium salt of H-D-iGlu-D-Trp-OH (1:1), characterized by peaks in the powder x-ray with the following 2θ values: 13,93, 20,02, 24,03 and 28,12.

17. Crystalline ammonium salt of H-D-iGlu-D-Trp-OH (1:1), characterized by a powder x-ray, which is shown in figure 2.

18. Crystalline ammonium salt of H-D-iGlu-D-Trp-OH (1:1), characterized by the following parameters of powder x-ray presented in values of interplanar distance d, Bragg angle 2θ and relative intensity (expressed as a percentage relative to the most intense line):

Angle °2θThe value of d, ARelative intensity [%]
9,299,5174,1
12,197,2584,5
13,936,35476,2
15,175,83727,4
16,495,3719,8
17,185,1573
18,564,77831,6
18,884,69610,5
20,024,431100
22,283,9863
23,313,8144,6
23,663,7579,8
24,033,752,9
24,373,64926,3
25,073,54911,4
25,613,4755,6
25,963,435
27,623,22729,7
28,123,1755,7
28,493,13112,2
29,523,02323,1
30,272,9513,7
30,642,9157,9
31,312,85411,8
31,72,82129,6
32,162,78119,2
32,812,72816
33,782,6527,4
34,142,6255,1
35,762,50916
36,942,431the 11.6
37,582,39125,6
38,032,36412,5
to 39.222,2951,9

19. Amorphous ammonium salt of D-isoglutamine-D-tryptophan (1:1).

20. Amorphous ammonium salt of D-isoglutamine-D-tryptophan (1:1), characterized by a powder x-ray, which is shown in figure 3.

21. The method of obtaining monoammonium salt of H-D-iGlu-D-Trp-OH, free from inorganic salts, comprising the following stages:
(a) obtaining a solution of an acid additive salt of H-D-iGlu-D-Trp-OH in aqueous medium essentially free of organic solvent;
(b) correction of the pH to values of from about 3.9 to about 8,6 with a solution of a metal hydroxide;
(c) treating the solution from step (b) ion exchange resin and elution with water for ion exchange of the metal salt in solution in hydrogen ion up until the pH of the eluate will not be from about 5.7 to about 7,0;
(d) contact ion-exchange resin with a regenerating solution on the basis of the ammonia used for the exchange of ions in it on the target H-D-iGlu-D-Trp-OH, contained in the ion-exchange resin, so that to form the eluate regenerating solution containing the ammonium salt of H-D-iGlu-D-Trp-OH; and
(e) evaporating the solvent from the solution from step (d) to obtain the crude ammonium salt;
and additionally comprising the following stages:
(f) dissolving the ammonium salt from step (e) in the water and slow addition of isopropanol so that a precipitate of monoammonium salt; and
(g) vacuum drying the product from step (f) to obtain the crystalline form of the ammonium salt of H-D-iGlu-D-Trp-OH (1:1); or
(h) processing of material from step (e) by chromatography on silica gel using a solution of isopropanol and ammonia as the eluent, and the removal of the isopropanol by solvent evaporation or dissolution of the product from step (g) in water to obtain a solution of ammonium salt of H-D-iGlu-D-Trp-OH (1:1) in water; and
(i) freeze drying the product from step (h) to obtain the amorphous form of the ammonium salt of H-D-iGlu-D-Trp-OH (1:1).

22. The method according to item 21, which use at least one solution of a metal hydroxide selected from sodium hydroxide solution, a solution of lithium hydroxide and potassium hydroxide solution.

23. The method according to item 21 or 22, in which the acid additive salt is a H-D-iGlu-D-Trp-OH hydrochloride obtained by the process comprising the following stages:
(i) basic hydrolysis of the compounds of formula I:
where R1selected from the group consisting of C1-C4of alkyl and benzyl, and R2represents a C1-C4alkyl, provided that C4alkyl is tert-bootrom,
using a metal hydroxide in water and an inert solvent, in the presence of methanol to obtain Boc-D-iGlu-D-Trp-OH, free of other diastereomers; and
(ii) removal using hydrogen chloride protection with Boc-D-iGlu-D-Trp-OH from the stage (i) in an inert organic solvent; and evaporating the solvent to obtain hydrochloride H-D-iGlu-D-Trp-OH.

24. The method according to item 21 or 22, in which the acid additive salt is a H-D-iGlu-D-Trp-OH hydrochloride, and the solution of acid additive salt at the stage of (a) receiving method including:
(i) hydrogenation of the compounds of formula II

where R1represents benzyl, and R2selected from the group consisting of benzyl, and hydrogen using palladium on coal in methanol or ethanol;
(ii) purification of crude H-D-iGlu-D-Trp-OH from the stage (i) using chromatography on silica gel using isopropanol and water as an eluent; and
(iii) processing of material from step (ii) chloroethanol acid in water to obtain a solution of hydrochloride salt of H-D-iGlu-D-Trp-OH in water.

25. The method according to item 23, in which R1represents benzyl, and R2represents methyl.

26. The method according to item 23, in which the inert solvent in stage (i) is selected from the group consisting of methanol and methyl tert-butyl ether; and a hydroxide of a metal selected from the group consisting of sodium hydroxide, lithium hydroxide and potassium hydroxide.

27. The method according to item 21 or 22, in which the acid additive salt on the stage (a) is a H-D-iGlu-D-Trp-OH hydrochloride, pH value corresponding to the predominant form of monovalent salt on the stage (b)is from about 5.7 to about 7,0, and ion-exchange resin at the stage (C) is an AMBERLYST®15.

28. The method of obtaining monoammonium salt of H-D-iGlu-D-Trp-OH crystalline H-D-iGlu-D-Trp-OH, free from inorganic salts, comprising the following stages:
(a) adding crystalline H-D-iGlu-D-Trp-OH for less than about one equivalent of a solution of ammonium hydroxide;
(b) correction of the pH to values in the range of from approximately 6 to approximately 7 using ammonium hydroxide;
(c) evaporating the solvent to obtain oil; adding isopropanol with stirring to cause precipitation of monoammonium salt;
(d) isolation of the precipitated ammonium salt of H-D-iGlu-D-Trp-OH; and
(e) vacuum drying the product obtained in stage (C), obtaining monoammonium salt of H-D-iGlu-D-Trp-OH.

29. The method of selection of crystalline H-D-iGlu-D-Trp-OH from a water solution of H-D-iGlu-D-Trp-OH is by using a graphical composition to determine the percentage of H-D-iGlu-D-Trp-OH against pH, where the percentage of forms H-D-iGlu-D-Trp-OH in solution is more than about 50% in solution with a pH from about 2.0 to about 3.2, and which leads to the precipitation of crystalline H-D-iGlu-D-Trp-OH.

30. The compound H-D-Glu-(γ-D-Trp-OR2)-α-OR1

and its pharmaceutically acceptable acid salt additive, where each of R1and R2independently selected from the group consisting of benzyl and C1-C4the alkyl.

31. Connection item 30, in which C1-C4alkyl selected from the group consisting of methyl, ethyl, isopropyl and tert-butyl.

32. Connection item 30 or 31, in which R1has the same meaning as R2.

33. Connection p, in which R1and R2represent methyl.

34. Connection p, in which R1and R2represent ethyl.

35. Connection p, in which R1and R2are isopropyl.

36. Connection p, in which R1and R2represents benzyl.

37. Connection p, in which R1and R2are tert-butyl.

38. Connection item 30 or 31, in which R1represents ethyl and R2represents methyl.

39. Connection item 30 or 31, in which R1represents ethyl and R2represents isopropyl.

40. Connect the tion under article 30 or 31, in which R1represents tert-butyl and R2represents isopropyl.

41. Connection item 30 or 31, in which R1represents benzyl and R2represents ethyl.

42. Connection item 30 or 31, in which R1represents benzyl or methyl and R2represents ethyl, isopropyl or tert-butyl.

43. Connection item 30 or 31, in which R1represents benzyl and R2represents methyl.

44. Pharmaceutical composition, which is an immunosuppressant or remedy of psoriasis containing a therapeutically effective amount of a compound according to any one of p-43 at least one pharmaceutically acceptable auxiliary substance.

45. The use of a therapeutically effective amount of a compound according to any one of p-43 as an immunosuppressant or anti-psoriasis.

46. Pharmaceutical composition, which is an immunosuppressant or remedy of psoriasis containing a therapeutically effective amount of amorphous monoammonium salt according to claim 19 or 20 and at least one pharmaceutically acceptable auxiliary substance.

47. Solid pharmaceutical composition, which is an immunosuppressant or remedy of psoriasis containing a therapeutically effective amount of crystalline is Oli on any of PP-18 and at least one pharmaceutically acceptable auxiliary substance.

48. The use of amorphous monoammonium salt according to claim 19 or 20 as an immunosuppressant or anti-psoriasis.

49. The use of a crystalline salt according to any one of p-18 as an immunosuppressant or anti-psoriasis.

50. Solid pharmaceutical composition which is an immunosuppressant or remedy of psoriasis containing a therapeutically effective amount of a crystalline salt of compound according to any one of claims 1 to 3 and at least one pharmaceutically acceptable auxiliary substance.

51. The use of crystalline compounds according to any one of claims 1 to 3 as an immunosuppressant or anti-psoriasis.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to organic chemistry, namely new 3,8-diaminotetrahydroquinoline derivatives of formula (1a) or to their pharmaceutically acceptable salts wherein X represents CH2, C=O or CH-OR; m is 1 or 2; Ar represents a phenyl group or a 5-merous or 6-merous aromatic heterocyclic group having one element specified in S and N, (wherein the phenyl group may be substituted by 1-2 halogen atoms); each R1 and R2 represents a hydrogen atom; R3 represents a C1-C6 alkyl group or indolyl-C1-4 alkyl group (the indolyl group is optionally substituted by a C1-C6 alkyl group or a halogen atom), n is 0; R4 and R5 which may be identical or different, each represents a hydrogen atom or a C1-C6 linear or branched alkyl group; each R6 and R7 represents a hydrogen atom; and R represents a hydrogen atom. Also, the present invention refers to a drug preparation and a pharmaceutical composition of the basis of the compound of formula (1a), to the compound of formula (F1), to a method for preparing an intermediate compound (e).

EFFECT: there are prepared new 3,8-diaminotetrahydroquinoline derivatives which possess high GHS-R antagonist activity.

10 cl, 1 tbl, 124 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to crystalline modifications: 1 (polymorphous form F), 2 (polymorphous form I) and 3 (polymorphous form X) of monosodium salt of D-isoglytamyl-D-tryptophan (1:1) characterised by powder X-ray pattern peaks presented in the application materials, as well as to pharmaceutical compositions containing them. The invention describes their use for treating various diseases and body conditions of at least one autoimmune diseases specified in a group consisting of psoriasis, atopic dermatitis and rheumatoid arthritis.

EFFECT: present invention describes the methods for producing the declared crystalline modifications of monosodium salt of D-isoglytamyl-D-tryptophan (1:1).

42 cl, 4 ex, 9 dwg

FIELD: chemistry.

SUBSTANCE: disclosed is a method of producing lipodipeptides based on L-glutamic acid or L-glutamine and L-ornithine, L-lysine or L-arginine. L-glutamic acid or L-glutamine derivatives esterified with fatty alcohol residues are obtained by fusing an amino acid with a corresponding alcohol in the presence of a strongly acidic ion-exchange resin in H+ form. Amino groups of L-ornithine, L-lysine or L-arginine are protected and then activated with carboxyl groups. Further, a reaction takes place between esterified derivatives of L-glutamic acid or L-glutamine and the protected derivatives of L-ornithine, L-lysine or L-arginine to form lipodipeptides. The protective groups are then removed.

EFFECT: invention simplifies the process at the esterification step, reduces reaction temperature and reaction time to 2 hours.

3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel polyfunctional fullerene C60 amino acid derivatives of formula (1) , wherein R is H, mono- or dinitroxyC1-6alkyl, maleinimide; N-Z denotes a α, β, γ, ω-amino acid fragment of general formula where m=2-5 and M is a nitroxyC1-6alkyl group, a C1-6alkyl group or an alkali metal salt, having biological activity, as well as methods for production thereof and a method for covalent bonding of fullerene derivatives with SH-containing proteins. The invention also relates to the use of nitroxyalkyl-N-(fullerenyl)amino acids as nitrogen monoxide donors and to use of nitroxyalkyl-N-(fullerenyl)amino acids as quick-acting vasodilatators for antihypertensive therapy. The invention also relates to a method of inhibiting a metastasis process and a method of enhancing antileukemic activity of cyclophosphamide. Disclosed nitroxyalkyl-N-fullerenyl amino acid derivatives have an effect on coronary, contractile and pumping ability of the isolated heart of Vistar rats and are quick-acting vasodilatators which reduce arterial pressure and heart rate and cause relaxation of coronary vessel with less depressive effect on myocardial function compared to nitroglycerine.

EFFECT: disclosed compounds considerably intensify antileukemic activity of cyclophosphamide, increase chemosensitising activity when combined with cyclophosphamide.

9 cl, 8 ex, 3 tbl, 3 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a 1-(1-adamantyl)ethylamine derivative (remantadin) of general formula: wherein R is a functional group of the amino acid residue (I-IV) or the lipoic acid residue (V). (I), (II), (III), (IV), (V) which possess selective antiviral activity in relation to the influenza A strains, including the viral strains resistant to action of remantadin. The compounds wherein R means the groups (l),(ll),(lll) and (V) are novel.

EFFECT: prepared compounds may be promising as substances as a part of the antiviral drugs and applicable for creating new antiviral drugs.

2 cl, 2 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: in claim described are organic compounds of formula I where radicals are given in description, which are applicable for elimination, prevention or alleviation of one or more symptoms, associated with HCV disorders.

EFFECT: obtaining pharmaceutical composition which possesses inhibiting activity with respect to NS3-4 HCV serinprotease, including formula I compound and pharmaceutically acceptable carrier.

30 cl, 25 ex, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to a compound of formula I

and pharmaceutical compositions containing them and used for IAP-function modulation by inhibiting the binding of BIR-binding protein and BIR domain of IAP and for treating proliferative diseases such as cancer. What is also disclosed is a compound of formula I marked by a detectable tag and an affinity tag which is applicable as a probe for identifying the compounds bound with BIR domain of IAP.

EFFECT: preparing the compounds used for IAP-function modulation.

33 cl, 4 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to methods for synthesis of nonapeptide ethylamide, having strong LH-RH/FSH-RH activity, of formula pGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NH-C2H5·2AcOH (I), and intermediate compounds for synthesis thereof. The nonapeptide ethylamide is obtained via condensation of a C-terminal tetrapeptide of formula H-D-Ser(But)-Leu-Arg-Pro-NH-C2H5·HCl (II) with a dipeptide of formula: X-Ser-Tyr-OH (IV), where X is a protective group. The obtained N-substituted hexapeptide ethylamide of formula X-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NH-C2H5·HCl (III) is treated with an unblocking agent to remove the N-protective group, and then condensed with a tripeptide of formula pGlu-His-Trp-OH·HCl (V) and the end product is purified through chromatography and extracted in form of a monoacetate salt.

EFFECT: high output.

4 cl, 1 ex

FIELD: chemistry.

SUBSTANCE: synthesis is carried out using a liquid-phase technique via condensation of pentafluorophenyl ether of Nα-benzloxycarbonyl-Nε-tert-butoxycarbonyl-L-lysine with methyl ether of O-tert-butyl-L-threonine in the presence of N-methylmorpholine in ethyl acetate medium and followed by hydrogenation of the obtained compound with hydrogen in methyl alcohol using palladium hydroxide as a catalyst. Purification of the desired protected dipeptide is carried out using a salt with oxalic acid. The initial pentafluorophenyl ether is obtained from reaction of Nα-benzyloxycarbonyl-Nε-tert-butoxycarbonyl-L-lysine with pentafluorophenol and is then used without extraction. The disclosed method enables to obtain the desired product with output of over 96% and high degree of purity.

EFFECT: obtained protected dipeptide can be used to produce octreotide or analogues thereof, or other peptides containing a L-lysine-L-threonine fragment.

2 ex

FIELD: chemistry.

SUBSTANCE: invention describes a method of producing cyclic depsipeptides of formula (I) via intramolecular cyclisation.

EFFECT: improved method.

14 cl, 31 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to crystalline modifications: 1 (polymorphous form F), 2 (polymorphous form I) and 3 (polymorphous form X) of monosodium salt of D-isoglytamyl-D-tryptophan (1:1) characterised by powder X-ray pattern peaks presented in the application materials, as well as to pharmaceutical compositions containing them. The invention describes their use for treating various diseases and body conditions of at least one autoimmune diseases specified in a group consisting of psoriasis, atopic dermatitis and rheumatoid arthritis.

EFFECT: present invention describes the methods for producing the declared crystalline modifications of monosodium salt of D-isoglytamyl-D-tryptophan (1:1).

42 cl, 4 ex, 9 dwg

The invention relates to products derived from histamine and, in particular, the condensation products of histamine or methylsiloxanes histamine and amino acids, the method of their preparation and use as active principle in areas such as therapy and cosmetology, as well as the factor (agent), improving the stability of compositions used in therapy, cosmetology, agriculture and food industry (region)

The invention relates to medicine, namely to new peptide structures with immunomodulatory properties, and preparations on their basis

The invention relates to medicine, namely to compounds having immunomodulatory properties

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. A fibrin powder is prepared by the known method. Fibrin is recovered from a blood clot and transformed into powder. It is added with an aqueous solution of neutral catholyte having pH 9 or more, reduction-oxidation potential - 300 - 500 mV and containing the active ingredients O2, HO2, HO2., H2O2, H, OH', in ratio: fibrin powder 1.0 g per 1.5-2.0 ml of the aqueous solution of neutral catholyte. It is mixed to form a plastic mixture.

EFFECT: invention provides preparing the fibrin paste which is used to improve the reparative processes in the wound.

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to combinations of peptides in each case with the same sequence length (SEQL) which can be prepared in a stable reproducible quality and quantity of a mixture (A) containing a number of x amino acid with protected acid groups or a number of z peptides with the acid groups protected by the protective groups and the activated amino groups, with the amino acids in the mixture (A) found in a specific molar ratio, and a mixture (B), containing a number of y amino acids with the amino groups protected by the protective groups, with a molar ratio of the amino acids of the mixture (B) being the same as the molar ratio of the amino acids of the mixture (A), and the number x=y, and x is a figure from 11 to 18.

EFFECT: new combinations of the peptides are presented.

13 cl, 2 dwg, 1 ex

FIELD: biotechnologies.

SUBSTANCE: method includes the following stages: conservation of cells in presence of buffered 80-90% glycerine, collapse of cell shells by 3% triton X-100, extraction by increasing concentrations of salts: 0.14 M, 0.35 M; 2 M NaCl, 6 M guanidine hydrochloride with 0.1% β-mercaptoethanol, extraction of positively charged proteins from above fractions with the help of ion-exchange chromatography with amberlite IRC-50 in an interrupted gradient of guanidine hydrochloride: 6%, 8.9%, 10.6%, 13% on 0.1 M potassium-phosphate buffer pH 6.8 and detection of sites of sensitivity in them to Arg-X proteolysis.

EFFECT: invention may be used in analysis of molecular-genetic mechanisms of procaryote cell structure formation and role of protein components in their organisation, and also when studying features of genome remodelling, which is necessary for opening of paths of regulation mechanisms of macro- and microorganisms.

9 dwg, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry. A biomass of the gram-negative bacteria Salmonella typhi fam. Enterobacteriacea is prepared. A cell wall peptidoglycane (CWP) of the bacteria is recovered by biomass extraction in 45% aqueous phenol at temperature 70-90°C or in aqueous solution of ionic or non-ionic detergents at temperature 37-100°C. That is followed by preparative enzymatic hydrolysis for insoluble CWP cleavage with the use of lysozyme at pH 4.5 - 8.9 and temperature 10 - 37°C. Simultaneously, a pharmacologically acceptable mixture of substances is removed by dialysis from the reaction mixture with the use of semipermeable membranes for ultrafiltration with cut-off size up to 5 kDa. The mixture of substances is also recovered by means of column gel-chromatography, particularly preparative gel chromatography on Sephadex or TSK gel columns. The end product yield is 320 mg.

EFFECT: method enables producing the pharmacologically acceptable mixture of substances containing the following ingredients: β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-(L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid) (GMtri); β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-(L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine) (GMtetra); and GMtetra dimer (diGMtetra) wherein a bond of monomer residues of GMtetra is enabled by a carboxyl group of terminal D-alanine of one GMtetra residue and ω-aminogroup of meso-diaminopimelic acid of the other GMtetra residue with the coupled tetrapeptide resides positioned in various polysaccharide chains.

4 cl, 2 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry. A biomass of the gram-negative bacteria Salmonella typhi fam. Enterobacteriacea is prepared. A cell wall peptidoglycane (CWP) of the bacteria is recovered by biomass extraction in 45% aqueous phenol at temperature 70-90°C or in aqueous solution of ionic or non-ionic detergents at temperature 37-100°C. That is followed by preparative enzymatic hydrolysis for insoluble CWP cleavage with the use of lysozyme at pH 4.5 - 8.9 and temperature 10 - 37°C. Simultaneously, a pharmacologically acceptable mixture of substances is removed by dialysis from the reaction mixture with the use of semipermeable membranes for ultrafiltration with cut-off size up to 5 kDa. The mixture of substances is also recovered by means of column gel-chromatography, particularly preparative gel chromatography on Sephadex or TSK gel columns. The end product yield is 320 mg.

EFFECT: method enables producing the pharmacologically acceptable mixture of substances containing the following ingredients: β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-(L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid) (GMtri); β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-(L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine) (GMtetra); and GMtetra dimer (diGMtetra) wherein a bond of monomer residues of GMtetra is enabled by a carboxyl group of terminal D-alanine of one GMtetra residue and ω-aminogroup of meso-diaminopimelic acid of the other GMtetra residue with the coupled tetrapeptide resides positioned in various polysaccharide chains.

4 cl, 2 tbl, 3 ex

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